Lena Serrander
email: lena.serrander@medecine.unige.ch

Laetitia Cartier

Karen Bedard

Botond Banfi

Bernard Lardy

Olivier Plastre

Andrzej Sienkiewicz

Lászlo Fórró

Werner Schlegel

Karl-Heinz Krause

NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation

Keywords: NADPH oxidases, ROS, hydrogen peroxide, superoxide, NOX4, NOX4 inhibitors ROS, reactive oxygen species, NOX, NADPH oxidases, HEK cells, human embryonic kidney cells, TC, tetracycline,

NOX4 is an enigmatic member of the NOX family of ROS-generating NADPH oxidases.

NOX4 has a wide tissue distribution, but the physiological function and activation mechanisms are largely unknown, and its pharmacology poorly understood. We have generated cell lines expressing NOX4 upon tetracycline induction. Tetracycline induced a rapid increase in NOX4 mRNA (1 h) followed closely (2h) by a release of reactive oxygen species (ROS). Upon tetracycline withdrawal, NOX4 mRNA levels and ROS release decreased rapidly (<24h). In membrane preparations, NOX4 activity was selective for NADPH over NADH and did not require the addition of cytosol. The pharmacological profile of NOX4 was distinct from other NOX isoforms: diphenyl iodonium (DPI) and thioridazine efficiently inhibited the enzyme, while apocynin and gliotoxin, did not (IC50 > 100 µM). The pattern of NOX4-dependent ROS generation was unique: i) ROS release upon NOX4 induction was spontaneous without need for a stimulus, and ii) the type of ROS released from NOX4 expressing cells was hydrogen peroxide, while superoxide was almost undetectable.

Probes that allow detection of intracellular superoxide generation yielded differential results: DHE fluorescence and ACP ESR measurements did not detect any NOX4 signal, while a robust signal was observed with NBT. Thus, most likely NOX4 generates superoxide within an intracellular compartment that is accessible to NBT, but not to DHE and ACP. In conclusion, NOX4 has a distinct pharmacology and pattern of ROS generation. The close correlation between NOX4 mRNA and ROS generation might hint towards a function as inducible NOX isoform.

INTRODUCTION

NOX4 is a member of the NOX family of NADPH oxidases. These enzymes transport electrons from cytoplasmic NADPH across cell and organelle membranes to generate superoxide and down-stream reactive oxygen species (ROS). NOX enzymes have been shown to be involved in the regulation of a wide range of physiological functions, including cell death and survival, differentiation, proliferation, Ca 2+ signalling, gene expression, and migration (reviewed in [START_REF] Lambeth | NOX enzymes and the biology of reactive oxygen[END_REF][START_REF] Bedard | The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology[END_REF]).

NOX4 was first described in 2000 [START_REF] Geiszt | Identification of renox, an NAD(P)H oxidase in kidney[END_REF][START_REF] Shiose | A novel superoxide-producing NAD(P)H oxidase in kidney[END_REF]. The enzyme is highly expressed in kidney, particularly in the tubular system [START_REF] Geiszt | Identification of renox, an NAD(P)H oxidase in kidney[END_REF]. However NOX4 is also expressed in many other cell types including endothelial cells [START_REF] Ago | Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase[END_REF], osteoclasts [START_REF] Yang | A New Superoxide-generating Oxidase in Murine Osteoclasts[END_REF], smooth muscle cells [START_REF] Ellmark | The contribution of Nox4 to NADPH oxidase activity in mouse vascular smooth muscle[END_REF], fibroblasts [START_REF] Cucoranu | NAD(P)H Oxidase 4 Mediates Transforming Growth Factor-{beta}1-Induced Differentiation of Cardiac Fibroblasts Into Myofibroblasts[END_REF],

mesangial cells [START_REF] Gorin | Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells[END_REF], adipocytes [START_REF] Mahadev | The NAD(P)H oxidase homolog Nox4 modulates insulinstimulated generation of H2O2 and plays an integral role in insulin signal transduction[END_REF], pancreatic islets [START_REF] Uchizono | Expression of isoforms of NADPH oxidase components in rat pancreatic islets[END_REF] and embryonic stem cells [START_REF] Li | The NADPH oxidase NOX4 drives cardiac differentiation: Role in regulating cardiac transcription factors and MAP kinase activation[END_REF]. It has been suggested to be involved in stress signals in kidney [START_REF] Gorin | Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells[END_REF][START_REF] Gorin | Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney[END_REF] and smooth muscle cells [START_REF] Pedruzzi | NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells[END_REF], TGFβ-induced differentiation [START_REF] Cucoranu | NAD(P)H Oxidase 4 Mediates Transforming Growth Factor-{beta}1-Induced Differentiation of Cardiac Fibroblasts Into Myofibroblasts[END_REF][START_REF] Sturrock | Transforming Growth Factor {beta}1 Induces Nox 4 NAD(P)H Oxidase and Reactive Oxygen Species-Dependent Proliferation in Human Pulmonary Artery Smooth Muscle Cells[END_REF][START_REF] Hu | Reactive oxygen species production via NADPH oxidase mediates TGFbeta-induced cytoskeletal alterations in endothelial cells[END_REF], insulin signaling [START_REF] Mahadev | The NAD(P)H oxidase homolog Nox4 modulates insulinstimulated generation of H2O2 and plays an integral role in insulin signal transduction[END_REF], oxygen sensing [START_REF] Maranchie | Nox4 is critical for hypoxia-inducible factor 2-alpha transcriptional activity in von Hippel-Lindau-deficient renal cell carcinoma[END_REF][START_REF] Lee | NOX4 as an oxygen sensor to regulate TASK-1 activity[END_REF], cardiac differentiation [START_REF] Li | The NADPH oxidase NOX4 drives cardiac differentiation: Role in regulating cardiac transcription factors and MAP kinase activation[END_REF], and transcriptional regulation [START_REF] Brar | An NAD(P)H oxidase regulates growth and transcription in melanoma cells[END_REF][START_REF] Kuroda | The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells[END_REF].

NOX4 expression is increased by TGFβ stimulation in fibroblasts [START_REF] Cucoranu | NAD(P)H Oxidase 4 Mediates Transforming Growth Factor-{beta}1-Induced Differentiation of Cardiac Fibroblasts Into Myofibroblasts[END_REF][START_REF] Sturrock | Transforming Growth Factor {beta}1 Induces Nox 4 NAD(P)H Oxidase and Reactive Oxygen Species-Dependent Proliferation in Human Pulmonary Artery Smooth Muscle Cells[END_REF] as well as by angiotensin stimulation of mesangial cells in the kidney [START_REF] Gorin | Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells[END_REF]. These stimulations were paralleled with an increase in ROS generation suggesting that NOX4 activity is regulated, at least in part, at the transcriptional level.

While NOX1, NOX2, and NOX3 activity depends on the presence of activator or organizer subunits (NOXAs and NOXOs) [START_REF] Bedard | The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology[END_REF], NOX4 is active in cells that do not express such cytoplasmic subunits. NOX4 does require the membrane-associated subunit p22 phox for function [START_REF] Martyn | Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases[END_REF], however, as opposed to NOX1-3, it does not require the organizer subunitinteracting proline-rich domain of p22 phox [START_REF] Kawahara | Point mutations in the proline-rich region of p22phox are dominant inhibitors of Nox1-and Nox2-dependent reactive oxygen generation[END_REF]. Data concerning its dependence on the small GTPase Rac and on the need for cell stimulation are contradictory. While some indirect data raise the possibility of Rac involvement, reconstitution data in a variety of cell types suggest that NOX4 activity does not require Rac and is constitutive [START_REF] Martyn | Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases[END_REF][START_REF] Banfi | A Ca(2+)-activated NADPH oxidase in testis, spleen, and lymph nodes[END_REF][START_REF] Ambasta | Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase[END_REF]. This constitutive activity of NOX4 upon heterologous expression is an interesting, but experimentally challenging feature. It raises the possibility that NOX4 might be an inducible NOX isoform, regulated on the level of mRNA level, rather than through post-translational protein modifications. However, this feature also presents a challenge for the study of NOX4 due to the toxicity associated with sustained ROS generation, as evidenced by cellular senescence upon heterologous NOX4 expression [START_REF] Geiszt | Identification of renox, an NAD(P)H oxidase in kidney[END_REF][START_REF] Shiose | A novel superoxide-producing NAD(P)H oxidase in kidney[END_REF].

Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903 © 2007 Biochemical Society

In this study we report the generation of a cell line that expresses NOX4 upon tetracycline-induction. We use this system to study basic features of NOX4 activity and its unusual pattern of ROS generation.

EXPERIMENTAL Material

Dulbecco's modified Eagle Medium (DMEM), Hank's buffered salt solution, Gateway cloning system including pENTRY and DEST 30 vectors, superscript II, fetal calf serum, blasticidin, neomycin (G418, geneticin), tetracycline (TC), cloning primers, calcein, and amplex red were purchased from Invitrogen. RNeasy columns and DNAse were purchased from Qiagen. Scopoletin, horse radish peroxidase (HRP), luminol, cytochrome C, diphenylene iodonium chloride (DPI), thioridazine and apocynin were purchased from Sigma Aldrich.

Human kidney RNA was purchased from Clonetech.

Cloning of human NOX4

Reverse transcription was carried out on human kidney RNA with superscript II (Invitrogen) according to the manufacturer's instructions. The full-length human NOX4 was amplified by PCR using the following primers: NOX4_F: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATG GCTGTGTCCTGGAGG NOX4_R:

GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGCTGAAAGACTCTTTATTGTAT

TC

The PCR product was cloned into a p221 vector (Invitrogen) and transferred into a pDEST 30 vector carrying a tet-on CMV promoter using the Invitrogen Gateway cloning system, according to the manufacturer's instructions.

Generation of inducible and stable cell lines HEK293 cells over-expressing a tetracycline receptor (T-REx HEK, Invitrogen) were transfected with the NOX4-containing tet-on vector (NOX4-pDEST30) by the calcium phosphate method [START_REF] Chen | Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA[END_REF], and selected with 400 µg/ml neomycin (G418) starting 3 days after transfection. HEK293 cells stably expressing NOX4 and NOX5 respectively were obtained by transfection of pcDNA3.1 with the full-length murine NOX4 or full-length human NOX5β cDNA inserted with neomycin as resistance gene followed by clone selection after neomycin culture for 10-14 days [START_REF] Banfi | A Ca(2+)-activated NADPH oxidase in testis, spleen, and lymph nodes[END_REF].

Cell culture

All cells were cultured in DMEM (cat no 61965, Invitrogen), 4.5 g/l glucose, supplemented with 10% foetal calf serum and penicillin (100 u/ml) and streptomycin 100 µg/ml) at 37°C in air with 5% CO 2 . Selecting antibiotics, blasticidin (5 µg/ml) and neomycin (400 µg/ml), were maintained in transfected cell cultures throughout.

ROS measurements

Scopoletin, Amplex Red, Cytochrome C reduction and luminol Extracellular hydrogen peroxide was measured by either Scopoletin or Amplex Red.

Scopoletin measurements were performed as previously described scopoletin [START_REF] De La Harpe | A semi-automated micro-assay for H2O2 release by human blood monocytes and mouse peritoneal macrophages[END_REF], with minor modifications. Briefly, detatched cells (20 000) were plated in 0.2 mL volume of HBSS containing 20 µM scopoletin and 0.5 U/ml horseradish peroxidase, and where indicated, stimuli and/or inhibitors. The plate was kept at 37°C and fluorescence readings were taken every minute for 40-60 minutes with excitation and emission wavelengths of 350 nm and 460 nm respectively in a Victor3 Wallac or BMG Fluostar microplate reader. Amplex Red measurements were done according to manufacturers instructions, with minor modifications. Briefly, detatched cells (50 000) were plated in 0.2 mL volume of HBSS containing 20 µM Amplex Red and 0.1 U/ml horseradish peroxidase, and where indicated, stimuli and/or inhibitors. The plate was kept at 37°C and fluorescence readings were taken every minute for 40-60 minutes with excitation and emission wavelengths of 550 nm and 600 nm respectively in a BMG Fluostar microplate reader.

Extracellular superoxide was measured by either Cytochrome C reduction or luminol, as described previously, with minor modifications [START_REF] Banfi | A Ca(2+)-activated NADPH oxidase in testis, spleen, and lymph nodes[END_REF]. For Cytochrome C, detatched cells (20 000) were plated in 0.2 mL volume of HBSS containing 150 µM Cytochrome C, and where indicated, stimuli and/or inhibitors. The plate was kept at 37°C and readings of the absorbance at 550 nm were taken every minute for 40-60 minutes in a Victor3 Wallac or BMG Fluostar microplate reader.

For luminol, detatched cells (20 000) were plated in 0.

ROS measurements in membranes preparations

Membrane were prepared as described previously [START_REF] Banfi | Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5)[END_REF]. Protein concentration was determined with Bradford reagent using bovine serum albumin as a standard. ROS activity was determined with the amplex red method described above with 5 µg protein/96-well containing 25 µM amplex red and 0.1 U/ml HRP in HBSS (with Ca 2+ and Mg 2+ ).

Real time PCR

Total RNA was isolated according to manufacturer's instructions using RNeasy (Qiagen).

cDNA was synthesized from 0.5 µg RNA, using a mix of oligo-dT and random hexamers, with Superscript II reverse transcriptase (Invitrogen) according to manufacturer's instructions.

The cDNA equivalent to 5 ng of total RNA was robot-pipetted and PCR-amplified in a final volume of 10 µl in triplicates in an ABI PRISM 7700 detection system (PE-Applied Biosystems).

Primers for the reference genes TATA-box-binding protein (TBP), β-glucoronidase (GusB)

and Elongation factor-1 alpha 1(EEF1A1) (see probe and primer sequences in table I), were designed by Primer Express 2.0 (Applied Biosystems, Foster City, CA). NOX4 was detected with primers and probe from Assay-on-Demand® no Hs00276431 from Applied Biosystems.

The quantitative PCR experiments and analysis were done with the help of the Genomic platform, Geneva.

Statistics

Results are expressed as mean ± standard error of the mean (SEM) of 3-8 experiments performed independently. Paired Student's t-test or ANOVA were performed when indicated and p<0.05 were considered significant and marked * for p<0.01 with ** and p<0.001 with ***.

RESULTS

ROS generation in a tetracycline-inducible NOX4 system

Because NOX4 is constitutively active upon heterologous expression, we generated a tetracycline inducible system that allowed controlled NOX4 expression. We used the T-REx system which is based on the expression of a tet repressor that binds to a modified CMV promoter and thereby maintains the latter in an inactive state. Tetracycline binds to the tet

Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903

repressor removing it from the promoter and thereby permitting transcription. As the tetrepressor expressing HEK293 cells (T-REx cells) were established with a blasticidin resistance, blasticidin was continuously included in the culture medium to ensure high level expression of the tet repressor, which is important to minimize residual expression (leaking).

To generate inducible NOX4 cells, the T-REx cells were transfected with a vector construct carrying the full-length human NOX4 cDNA driven by the modified CMV promoter. After 14 days of neomycin selection, several clones were established and investigated for the release of ROS upon tetracycline-induction using a scopoletin assay.

Among 6 clones tested, the increase in the rate of ROS production upon TC induction ranged from no change to 161 times that of un-induced cells (data not shown). Fig. 1a shows the clone selected for further experiments. In the absence of tetracycline, there is no detectable release of ROS into the medium. Tetracycline did not lead to ROS release in non-transfected cells (i.e. cells expressing the tet repressor only), nor did it enhance ROS release in cells stably expressing NOX4 (Fig. 1b).

Quantitative analysis of ROS release using the amplex red technique yielded the following results: non-transfected HEK T-REx cells generated less than 0.2 fmoles

H 2 O 2 /cell/h. Non-induced NOX4 T-REx HEK cells produced 1.5±0.8 fmoles H 2 O 2 /cell/h, while tetracycline-induced cells (1µg/ml, 24h) generated 83±24 fmoles H 2 O 2 /cell/h after a 24
hour TC induction (n=6). To put these numbers into context, tetracycline-induced NOX4 cells released roughly equal amounts of H 2 O 2 /cell as a reconstituted NOX2 system in HEK cells (data not shown) or NOX5-expressing HEK cells (see below), but approximately a 5-10 times less than PMA-stimulated neutrophils (data not shown). Thus, the capacity of NOX4 is probably in a similar range to that of other NOX enzymes.

We next investigated the effect of tetracycline concentration in the system. Cells were induced with tetracycline concentrations ranging from 1.25 fg/ml to 10 µg/ml and NOX4 activity was assessed by ROS generation (Fig. 2). In the absence of tetracycline, background ROS generation was below 3% of that in cells induced with 1 µg/ml TC. The EC 50 for tetracycline was 91 pg/ml. We chose a concentration of 1 µg/ml for further experiments since this concentration was well within the plateau range without apparent toxic effects. In order to investigate the reversibility of the NOX4 induction, we followed NOX4 mRNA levels and ROS generation after tetracycline withdrawal. NOX4 mRNA levels decreased by more than 50% after 4 hours, while a 50% decrease in ROS required more than 8 hours (Fig 3 c andd). In contrast, two other NOX2 inhibitors, which inhibit NOX2 in the low micromolar range inhibited NOX4 only with a very low affinity. Apocynin, which inhibits neutrophil ROS generation with an IC50 of approximately 10 µM [START_REF] Van Den Worm | Effects of methoxylation of apocynin and analogs on the inhibition of reactive oxygen species production by stimulated human neutrophils[END_REF], had an IC50 of >200 µM for NOX4. Gliotoxin, an aspergillus fumigatus toxin inhibits NOX2 with an IC 50 of 5-10 µM [START_REF] Tsunawaki | Fungal metabolite gliotoxin inhibits assembly of the human respiratory burst NADPH oxidase[END_REF][START_REF] Nishida | Fungal metabolite gliotoxin targets flavocytochrome b558 in the activation of the human neutrophil NADPH oxidase[END_REF]. The effect of gliotoxin on NOX4 showed some low efficacy inhibition, but the Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903 © 2007 Biochemical Society maximal inhibition achieved was around 40% with 100 µM gliotoxin therefore IC50 values could not be calculated. Thus, NOX4 has a distinct profile of inhibition as compared to NOX2 (table II).

Characteristics of NOX4-dependent ROS generation

When different techniques to measure ROS release from cells were applied, we found an unusual pattern in NOX4-expressing cells. NOX4 activity induced a strong signal with probes that detect extracellular hydrogen peroxide, such as scopoletin (Fig. 12) and amplex red (Fig. 3, 6a and b), but not with probes that detect extracellular superoxide, such as cytochrome C reduction (Fig. 6c,d) and horseradish peroxidase-dependent luminol-enhanced chemiluminescence (Fig. 6e,f). This contrasts with the behaviour of other NOX enzymes, such as NOX5, which yields a strong signal with probes detecting superoxide (Fig 6, c-f).

Note that in these studies, PMA-stimulated NOX5 activation was used; the PMA effects on NOX5-expressing cells have characteristics comparable to the previously described ionomycin effects (data not shown and [START_REF] Jagnandan | Novel Mechanism of Activation of NADPH Oxidase 5: CALCIUM SENSITIZATION VIA PHOSPHORYLATION[END_REF]). Note also that NOX4 activity is independent of or even slightly inhibited by PMA (Fig 6b).

Our results suggest that NOX4 either directly generates hydrogen peroxide or that it generates intracellular superoxide which dismutates intracellularly to the membrane permeant hydrogen peroxide. To distinguish between these possibilities, we used techniques to detect intracellular superoxide. The dihydroethidium (DHE)-method has been widely used to detect intracellular superoxide generation. Indeed, the non-fluorescent, membrane-permeant DHE enters cells freely. Upon interaction with superoxide, the membrane impermeant ethidium cation is liberated, which becomes fluorescent upon intercalation DNA. As shown in Fig. 7, no NOX4 signal was detected with the DHE method, while there was a clear NOX5 signal.

These results are interesting, but they do not globally exclude an intracellular superoxide generation by NOX4. Indeed a recent study demonstrated that the DHE method does not allow detection of superoxide generated by NOX2 in the phagosome [START_REF] Rinaldi | Evaluation of assays for the measurement of bovine neutrophil reactive oxygen species[END_REF].

We next investigated NOX4-dependent ROS generation using electron spin resonance (ESR). With appropriate spin trap agents, ESR can specifically detect superoxide. ACP (1-Acetoxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine) is an ESR spin trap agent that has been shown to enter cells in its ester form which is then hydrolyzed by cytosolic esterases. This hydrolyzed form is able to interact with intracellular superoxide [START_REF] Takeshita | In vivo monitoring of hydroxyl radical generation caused by xray irradiation of rats using the spin trapping/EPR technique[END_REF][START_REF] Yokoyama | In vivo EPR imaging by using an acyl-protected hydroxylamine to analyze intracerebral oxidative stress in rats after epileptic seizures[END_REF]. Thus, ACP is thought to be an appropriate spin trap agent to measure intracellular superoxide generation. Thus, the DHE and the ESR results reasonably exclude NOX4-dependent superoxide generation in the cytosol, and probably also in the nucleus and inner mitochondrial space, but they do not exclude the possibility that NOX4-dependent superoxide generation takes place within some other intracellular organelle.

One of the oldest and most established methods to detect intracellular superoxide generation is the reduction of nitro blue tetrazolium (NBT) to formazan, a dark blue precipitate. As NBT needs to be reduced, only superoxide (which may act as electron donor or acceptor), but not hydrogen peroxide (which is exclusively an oxidizing agent) is capable of reacting with NBT. Furthermore, NBT preferentially detects intracellular superoxide [START_REF] Berridge | Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction[END_REF]. As assessed by a quantitative NBT test (Fig. 9), tetracycline-induced NOX4 cells elicited a significant NBT reduction, as compared to non-induced cells. This signal was completely blocked by DPI (5uM). The NOX4-dependent NBT signal is a good argument in favor of superoxide generation. We can however not formally exclude a diaphorase activity (i.e. NBT would receive electrons directly from NOX4 without a superoxide intermediate). We also analyzed the subcellular distribution of formazan precipitates by light microscopy. No formazan precipitates were observed in uninduced cells (Fig. 9a). In tetracycline-induced cells (Fig. 9b), the precipitates showed a punctuate staining within the cytoplasm. In many cells, this staining was in the perinuclear region, but occasionally also in the cell periphery. The nucleus was systematically spared, but the nuclear envelope (which is part of the endoplasmic reticulum) showed a strong staining.

Discussion

In this study, an inducible cell line was used to analyze basic biochemical properties of the NOX family NADPH oxidase NOX4. Given the fact that NOX4 generates substantial amounts of ROS without a need for cell stimulation [START_REF] Geiszt | Identification of renox, an NAD(P)H oxidase in kidney[END_REF][START_REF] Shiose | A novel superoxide-producing NAD(P)H oxidase in kidney[END_REF][START_REF] Martyn | Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases[END_REF], a stable expression system has inherent dangers: i) damage to DNA which could lead to cellular senescence [START_REF] Geiszt | Identification of renox, an NAD(P)H oxidase in kidney[END_REF] or -in cells with deficient p53 function (e.g. HEK293 cells) -to an accumulation of DNA mutations; ii) damage to mitochondria which may lead to an increased mitochondrial ROS generation; and iii) permanent upregulation of antioxidant responsive genes, which would make it difficult to distinguish the acute NOX4 effects from a long-term antioxidant response. Thus, Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903 © 2007 Biochemical Society an inducible NOX4 system, as described in this manuscript, is preferable to cells stably expressing NOX4.

An additional advantage of the inducible system is that it offers the possibility to study the temporal relationship between mRNA levels and enzyme function. And one of the striking results of our study is that NOX4 mRNA levels are closely followed by NOX4 activity.

Unfortunately, there are presently no antibodies reliably recognizing human NOX4. Thus, it is presently not possible to calculate protein half-life. However, our results based on NOX4 function suggest that the NOX4 protein is rapidly (i.e. within a time frame of hours) synthesized and degraded. Note that this is in stark contrast to available data on NOX2, where the protein and its function persist for several days after transcriptional activity is shut down [START_REF] Borregaard | Development of neutrophil granule diversity[END_REF]. Obviously, a heterologous expression system cannot determine whether NOX4 is regulated on a transcriptional level [START_REF] Cucoranu | NAD(P)H Oxidase 4 Mediates Transforming Growth Factor-{beta}1-Induced Differentiation of Cardiac Fibroblasts Into Myofibroblasts[END_REF][START_REF] Sturrock | Transforming Growth Factor {beta}1 Induces Nox 4 NAD(P)H Oxidase and Reactive Oxygen Species-Dependent Proliferation in Human Pulmonary Artery Smooth Muscle Cells[END_REF] in endogenously NOX4 expressing cells. However our results clearly demonstrate that NOX4 has the potential to function as an inducible "iNOX".

To our knowledge, this is the first study of NOX4 activity in a broken cell system. It unequivocally demonstrates that NOX4 is functional in the absence of cytosol, reinforcing the concept that NOX4 activity is independent of cytosolic subunits. Importantly, this system also allowed the study of the NOX4 electron source. It is now clear that NOX2, NOX5, and NOX1 specifically use NADPH, and not NADH, as electron donor (reviewed in [START_REF] Bedard | The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology[END_REF]). However, it has been suggested that ROS-generating enzymes, in the vascular system, might prefer NADH over NADPH [START_REF] Griendling | Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells[END_REF] and for that reason several authors still use the term "NAD(P)H oxidase".

Our study reveals that NOX4 is indeed an NADPH oxidase with a preference for NADPH over NADH. Thus, we suggest that the term NAD(P)H oxidase should no longer be used when refering to NOX enzymes.

The inhibitor analysis shown in this study is, to our understanding, a first pharmacological profiling of NOX4. Not unexpectedly DPI, a large spectrum inhibitor of electron transporters including various NOX enzymes, mitochondrial oxidase, and xanthine oxidase (reviewed in [START_REF] Bedard | The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology[END_REF]) was found to inhibit NOX4. The following arguments suggest that the DPI effect is not due mitochondrial inhibition: i) inhibition of ROS generation occured in the submicromolar range, while inhibition of mitochondrial oxidase occurs with an IC50 of approximately 10 µM DPI [START_REF] Hool | Role of NAD(P)H oxidase in the regulation of cardiac L-type Ca2+ channel function during acute hypoxia[END_REF][START_REF] Li | Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production[END_REF]; ii) no ROS generation was detected unless NOX4 expression was induced by tetracycline. Thioridazine, another efficient NOX4 inhibitor, lacks specificity, as it also inhibits mitochondrial oxidase and xanthine oxidase in an almost identical concentration range [START_REF] Rodrigues | Thioridazine interacts with the membrane of mitochondria acquiring antioxidant activity toward apoptosis--potentially implicated mechanisms[END_REF][START_REF] Hadjimitova | Effect of some psychotropic drugs on luminol-dependent chemiluminescence induced by O2-, *OH, HOCl[END_REF] © 2007 Biochemical Society NOX4 inhibitors. In the case of apocynin, this is expected as apocynin is thought i) to inhibit NOX2 subunit assembly and ii) to require myeloperoxidase-dependent metabolism for full activity [START_REF] Stolk | Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol[END_REF]. The mode of action of gliotoxin is poorly understood, but clearly it is not a useful NOX4 inhibitor. In conclusion, NOX4 displays a unique pharmacological profile, distinct from that of NOX2 (table II).

The atypical pattern of NOX4-dependent ROS generation identified in this study is most compatible with a NOX4-dependent superoxide generation within a tight intracellular compartment, devoid of DNA. The alternative explanation, namely a direct hydrogen peroxide generation by NOX4 appears less likely since: i) NOX enzymes are mono-electron transporters that transport single electrons across a membrane (reviewed in [START_REF] Bedard | The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology[END_REF]) ii) hydrogen peroxide does not reduce NBT and there could not account for the observed NBT signal. This conclusion is also in line with a recent study analysing ROS generation in smooth muscle cell membranes [START_REF] Clempus | Nox4 is required for maintenance of the differentiated vascular smooth muscle cell phenotype[END_REF]. Localization studies of NOX4 are notoriously difficult. Antibody studies suggest a localization close to focal adhesions, within the nucleus or in the endoplasmic reticulum [START_REF] Kuroda | The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells[END_REF][START_REF] Martyn | Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases[END_REF][START_REF] Ambasta | Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase[END_REF]. However, the specificity of currently available NOX4 antibodies is not undisputed. The use of tagged NOX4 is also not without pitfalls, as it is hard to exclude the possibility that the tag alters cellular localization. In addition, C-terminal tags impair NOX4 activity (unpublished data and [START_REF] Martyn | Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases[END_REF]). With this limitation in mind, N-terminally eGFP-tagged NOX4 yielded interesting results: fluorescence displayed a punctuate pattern consistent with intracellular membranes, possibly the endoplasmic reticulum [START_REF] Martyn | Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases[END_REF][START_REF] Van Buul | Expression and localization of NOX2 and NOX4 in primary human endothelial cells[END_REF]. Our data also support the hypothesis that NOX4 is localized in intracellular membranes. The subcellular localization of NBT precipitation is thought to reflect at least to some extent the site of superoxide generation as the reduced NBT is water-insoluble and precipitates rapidly [START_REF] Berridge | Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction[END_REF]. Thus, the punctate localization of the formazan precipitations, in particular around the nuclear envelope (Fig. 8) is consistent with previous studies which report NOX4 localization in the ER. An ER localization is consistent with the DHE findings. While DHE can freely cross cell membranes, the ethidium cation generated after reduction by superoxide is impermeant. Thus failure of DHE to detect a signal suggests that superoxide is generated within subcelluar compartment devoid of nucleic acids, such as the ER.

Fundamentally, there are at least two possible theories to explain the physiological need for a superoxide-producing enzyme within intracellular membranes. NOX4 might regulate the redox environment within an intracellular organelle; this would be particularly pertinent for the endoplasmic reticulum which is supposed to maintain an oxidizing environment in order to assure proper posttranslational processing of proteins.

Time

  course of induction of mRNA and functional activity We investigated the time course of NOX4 induction by tetracycline. Both, NOX4 mRNA and ROS production showed a time-dependent increase (Fig 3a and b). However mRNA Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903 elevations were detected earlier than the increased ROS generation (1 and 2 hours after tetracycline addition, respectively). The fact that ROS generation was detectable after 2 hours suggests a relatively rapid transcriptional and translational processing of NOX4. Longer times of tetracycline induction resulted in a slight reduction of activity possibly reflecting cell toxicity (data not shown).

  NOX4 activity in membrane fractions: independence of cytosol and selectivity for NADPH over NADH We next investigated ROS generation in membrane fractions without added cytosol using amplex red. As shown in Figure 4, a strong signal was only observed with NADPH as electron donor. The signal with NADH was only slightly above background values, demonstrating that NOX4 preferentially uses NADPH. Thus NOX4 is similar to other members of the NOX family, a true NADPH oxidase. No enhancement of activity was obtained by addition of cytosol (data not shown), confirming that NOX4 activity is independent on cytosolic subunits for function. Thus we conclude that NOX4 is, similar to other members of the NOX family, selective for NADPH over NADH.Pharmacological profile of NOX4 activityIn order to characterize potential inhibitors, four known NOX2 inhibitors were tested for an effect on NOX4 activity (Fig5). DPI, the most commonly used agent to block NOX activity, and thioridazine, an anti-psychotic drug that inhibits NOX2 activity in neutrophils[START_REF] Traykov | Effect of phenothiazines on activated macrophage-induced luminol-dependent chemiluminescence[END_REF], inhibited NOX4 with high to intermediate affinity, in a concentration range similar to that observed for NOX2. DPI blocked NOX4 activity at concentrations in the submicromolar range (IC50 of 0.2 µM), while thioridazine blocked at low micromolar concentrations (4 µM).

  NOX4-activity did not lead to a detectable ESR signal, whereas significant signals could be Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903 © 2007 Biochemical Society detected in PMA-stimulated NOX5 cells (Fig 8). However, the design of ACP (i.e. hydrolysis by cytosolic esterases) makes it suited to detect of cytoplasmic superoxide generation, and not for measuring superoxide generation within intracellular organelles.

  NOX4 might Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903 © 2007 Biochemical Society however also regulate protein oxidation in the region surrounding the organelle through diffusion of hydrogen peroxide. In this case, NOX4 might generate signals including regulation of activity of kinases, phosphatases and transcription factors.

Figure 1 ROS

 1 Figure 1

Figure 2 Induction

 2 Figure 2

Figure 3 BiochemicalFigure 4 NOX4

 34 Figure 3

Figure 5 EffectFigure 6 Hydrogen

 56 Figure 5

Figure 7 DihydroethidiumFigure 8 ESR

 78 Figure 7

Figure 9 Intracellular

 9 Figure 9

Figure 2 Tetracycline

 2 Figure 2

2 mL volume of HBSS containing Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903 10

  µM luminol, 0.5 U/ml HRP, and where indicated, stimuli and/or inhibitors. The plate was kept at 37°C and light emission was recorded continuously using a Victor3 Wallac or a BMG Fluostar microplate reader. Cells were plated in 12 well plates and in the case of T-REx NOX4, induced with TC, 24 prior to experiments. Cells were washed in HBSS and incubated with NBT (1.6 mg/ml) in HBSS at 37°C for 45 min. After fixation in 100% methanol and a wash in methanol, the formazan precipitates were dissolved by addition of 560 µl 2M KOH and 480 µl DMSO. The amount of reduced NBT was quantified by determination of OD at 630 nm. Photographs were taken before fixation, using a Nikon Coolpix CCD camera (Nikon) mounted on an Axiovert S100 microscope (Zeiss) using a 100X objective. Electron spin resonance (ESR): Cells were detached and resuspended in HBSS (supplemented with Ca 2+ and Mg 2+ ) to 1 ×10 7 /ml and incubated with 100 µM ACP for 45 min at 37°C. For performing ESR measurements, aliquots of approximately 12 µl were transferred into 1.0 mm ID /1.2 mm OD quartz capillary tubes from VitroCom, NJ, USA (sample height of 50 mm) and sealed on both ends with Cha-Seal TM tube sealing compound (Medex International, Inc.,

	Dihydroethidium (DHE): Cells were plated in 96 well plates and in the case of NOX4,
	induced with TC, 24 prior to experiments. DHE (5 µM) was added to each well 5 min before
	addition of stimuli. Fluorescence (490nm/600nm, ex/em) was recorded during 60 min, once
	per minute in a Fluostar microplate reader at 37°C.
	Nitro blue tetrazolium (NBT):

USA). ESR experiments were carried out at room temperature using an ESP300E spectrometer (Bruker BioSpin GmbH, Karlsruhe, Germany), which was equipped with a standard rectangular mode TE 102 cavity. Instrument settings were: microwave power 20mW, microwave frequency 9.775 GHz, magnetic field 3480±60 G, modulation frequency 100 kHz, modulation

  amplitude 1.045 G, receiver gain 2 x 10 3 , time constant 20.48 ms, conversion time 40.96 ms, and total scan time 41.9 s. Five-scan field-swept spectra were accumulated for each ESR trace.Cell numbers were determined by calcein fluorescence in order to calculate the amount of hydrogen peroxide produced/cells using 490 nm excitation and 520 nm emission filters in separate wells after 10 min incubation with calcein (2.5 µM) at 37°C and normalized to a standard curve of counted cell numbers.

	Quantification of cell numbers

Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903

Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903

  . In contrast, neither apocynin nor gliotoxin are efficient

Table titles Table I Primers

 titlesI for quantitative PCR

Table II A

 II comparison of the effect on various inhibitors on NOX4 as well as on NOX2 and mitochondrial ROS generation

Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903

Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903 © 2007 Biochemical Society

			Table I				Figure 1
	H 2 O 2 production	(∆scopoletin fluorescence, RLU)	primer 70000 Hs-TBP_for Hs-TBP_rev Hs-GusB_for Hs-GusB_rev Hs-EEF1A1_for Hs-EEF1A1_rev Probe Hs-TBP_T Hs-GusB_T Hs-EEF1A1_T 0 10000 20000 30000 40000 50000 cl 20 + TC Sequence GCCCGAAACGCCGAATATA CGTGGCTCTCTTATCCTCATGA CCACCAGGGACCATCCAAT AGTCAAAATATGTGTTCTGGACAAAGTAA AGCAAAAATGACCCACCAATG GGCCTGGATGGTTCAGGATA CCGCAGCAAACCGCTTGGGA CCTGACTGACACCTCCAAGTATCCCAAGG CACCTGAGCAGTGAAGCCAGCTGCTT 60000 cl 20 a
			0	10	20	30	40	50
			Table II	Time (min)
			Inhibitor		NOX4 IC50	NOX2 IC50	mitochondrial oxidase IC50
						µM	µM	µM
			DPI	.2			0.9 [46]	5-10 [39]
	H 2 O 2 production	(∆scopoletin fluorescence, RLU)	Thioridazine Apocynin Gliotoxin 0 20000 40000 60000	5 215 > 100 stable NOX4+TC WT TRex WT TREX + TC stable NOX4 b	0.2-0.5 [47] 0.5-2 [28] 10 [29] 3-10 [30]	10 [41] > 30 [48] > 300 [39] 0.3 [49]
			0	10	20	30	40	50
				Time (min)

Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903

Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903

Acknowledgements

We would like to thank the collaborators from the genomics platform of the NCCR, Geneva for assistance with the design of the quantitative PCR experiments, Bianca Mottironi for helpful discussions and Marie-Claude Jaquot for technical assistance. We thank Katarzyna Pierzchała for kind assistance in performing ESR measurements. Vincent Jaquet and Sten Theander are acknowledged for their careful reading of the manuscript. This work was supported by grants from the Swiss National Science Foundations: MHV grant (LS) and project grant 3100A0-103725 (KHK). Biochemical Journal Immediate Publication. Published on 15 May 2007 as manuscript BJ20061903


Dario Bonanomi

Laura Rusconi

Agnese Chiara

Colombo

Fabio Benfenati

Flavia Valtorta
email: valtorta.flavia@hsr.it

Dibit 3a3

Synaptophysin I selectively specifies the exocytic pathway of Synaptobrevin2/VAMP2

Keywords: trafficking, protein interactions, exocytosis, endocytosis, endosomes

Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In this study we address the targeting of synaptobrevin2/vesicle associated membrane protein 2 (VAMP2), a critical component of the synaptic vesicle fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically-expressed synaptophysin I interacts with VAMP2 and converts its default surface targeting to a prominent vesicular distribution, with no effects on the targeting of other membrane proteins. Protein-protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the carboxyterminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles prior to surface delivery, without influencing VAMP2 endocytosis. Consistently, dynamin and alpha-SNAP mutants which block trafficking at the plasma membrane do not occlude the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.

INTRODUCTION

Communication in the nervous system relies on exocytosis of neurotransmitter-filled synaptic vesicles (SVs) that release their content at synapses. SVs are locally regenerated in the nerve terminals following exocytosis, whereas de novo formation of SVs requires delivery of new constituents from the cell body to synapses. Collapse of SVs with the plasma membrane during exocytosis is followed by endocytosis through clathrin-coated vesicles which fuse with endosomal compartments, from which SVs are regenerated [START_REF] Bonanomi | Protein sorting in the synaptic vesicle life cycle[END_REF]. Thus, segregation of SV proteins from either plasma membrane or endosomal resident proteins needs to take place in order to preserve the molecular identity of SVs [START_REF] Bonanomi | Protein sorting in the synaptic vesicle life cycle[END_REF][START_REF] Hannah | Synaptic vesicle biogenesis[END_REF] Since at least some of the SV proteins are delivered to synapses in distinct membrane carriers, assembly of SVs might proceed through the stepwise sorting of individual or groups of components onto the nascent organelle at the level of either endosomal compartments or the plasmalemma [1]. Importantly, SV proteins do not display common motifs that dictate their trafficking during SV biogenesis and upon expression in non-neuronal cells they segregate in distinct subcellular compartments [START_REF] Feany | The synaptic vesicle proteins SV2, synaptotagmin and synaptophysin are sorted to separate cellular compartments in CHO fibroblasts[END_REF].

The complexity of targeting information of SV proteins prompted the question as to whether a precise hierarchy of sorting determinants could operate during assembly of SVs. Although the molecular mechanisms underlying the sorting of SV proteins from resident components of the donor membranes are as yet unclear, protein-protein and protein-lipid interactions are likely to play a key role in these processes [START_REF] Bonanomi | Protein sorting in the synaptic vesicle life cycle[END_REF][START_REF] Hannah | Synaptic vesicle biogenesis[END_REF]. Thus, the sorting information might be present in a subset of SV proteins with the other components being secondarily targeted to SVs by association with the former proteins.

Owing to the essential function of the v-SNARE (vesicle soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) synaptobrevin2/VAMP2 in SV fusion [START_REF] Schiavo | Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin[END_REF][START_REF] Schoch | SNARE function analyzed in synaptobrevin/VAMP knockout mice[END_REF] and retrieval [START_REF] Deak | Synaptobrevin is essential for fast synaptic-vesicle endocytosis[END_REF] the sorting of this protein to SVs has been extensively investigated. Endocytosis and sorting from endosomal compartments are required for axonal polarization and targeting to SVs of VAMP2 [START_REF] Grote | A targeting signal in VAMP regulating transport to synaptic vesicles[END_REF][START_REF] Grote | Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal[END_REF][START_REF] West | Targeting of the synaptic vesicle protein synaptobrevin in the axon of cultured hippocampal neurons: Evidence for two distinct sorting steps[END_REF][10]. Targeting of VAMP2 to presynaptic sites is controlled by synaptophysin I, a major tetraspan SV membrane protein [START_REF] Pennuto | Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles[END_REF][START_REF] Valtorta | Synaptophysin: Leading actor or walk-on role in synaptic vesicle exocytosis?[END_REF]. The role of synaptophysin I in the SV life cycle is still unclear, mainly because of the absence of major phenotypic changes in synaptophysin I deletion mutants [START_REF] Eshkind | Mice lacking synaptophysin reproduce and form typical synaptic vesicles[END_REF][START_REF] Mcmahon | Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release[END_REF][START_REF] Abraham | Synaptic tetraspan vesicle membrane proteins are conserved but not needed for synaptogenesis and neuronal function in caenorhabditis elegans[END_REF], although a contribution of the protein to neurotransmitter release, synapse formation and SV endocytosis has been proposed [START_REF] Valtorta | Synaptophysin: Leading actor or walk-on role in synaptic vesicle exocytosis?[END_REF].

On the SV membrane VAMP2 is engaged in a complex with synaptophysin I, which is mutually exclusive with the formation of fusogenic SNARE complexes [START_REF] Calakos | Vesicle-associated membrane protein and synaptophysin are associated on the synaptic vesicle[END_REF][START_REF] Washbourne | Vesicle-associated membrane protein-2 (synaptobrevin-2) forms a complex with synaptophysin[END_REF][START_REF] Edelmann | Synaptobrevin binding to synaptophysin: A potential mechanism for controlling the exocytotic fusion machine[END_REF]. Consistently, stimulationinduced release of VAMP2 from synaptophysin I precedes SV fusion and makes SVs competent for exocytosis [19][START_REF] Pennuto | Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses[END_REF][START_REF] Reisinger | The synaptophysin/synaptobrevin complex dissociates independently of neuroexocytosis[END_REF][START_REF] Bonanomi | Taipoxin induces synaptic vesicle exocytosis and disrupts the interaction of synaptophysin I with VAMP2[END_REF]. The formation of hetero-complexes also underlies the ability of synaptophysin I to regulate the targeting of VAMP2 to SVs [START_REF] Pennuto | Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles[END_REF].

In order to define at which step of the exo-endocytic pathway synaptophysin I recruits newly synthesized VAMP2 to SV precursors and to determine the minimal requirements for the ability of synaptophysin I to direct the sorting of VAMP2, we reconstituted this process in non-neuronal cells allowing us to investigate the intrinsic determinants of VAMP2 sorting in the absence of confounding effects from other neuron-specific molecules. We show that, at early stages along the secretory pathway, synaptophysin I directs sorting of VAMP2 to vesicles exhibiting limited availability for constitutive exocytosis.

MATERIALS AND METHODS

Materials

The following primary antibodies were used: mouse anti-synaptophysin I, anti-VAMP2, antiα/βSNAP (Synaptic Systems, Göttingen, Germany); mouse anti-green fluorescent protein (GFP) (for western blotting) and mouse anti-hemagglutinin (HA) (Roche Diagnostics, Indianapolis, IN); mouse anti-GFP (3E6) (for immunofluorescence) (Quantum Biotechnologies, Montreal, Canada) [both anti-GFP (anti-FP) antibodies recognize the spectral variants yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)]; mouse anti-transferrin receptor (TfR) (Zymed, San Francisco, CA). Texas Red and FITC-conjugated secondary antibodies and pure rabbit IgG antibodies were from Jackson ImmunoResearch (West Grove, PA). Peroxidaseconjugated anti-rabbit was from Bio-Rad (Hercules, CA). The enhanced chemiluminescence detection system was from Amersham Bioscience (Little Chalfont Buchinghamshire, UK) and BCA protein assay reagent was from Pierce Chemical (Rockford, IL). FITC-conjugated transferrin were from Molecular Probes (Eugene, OR).

DNA contructs

Expression plasmids for enhanced CFP (ECFP), enhanced YFP (EYFP), SypI-CFP, SypI-YFP, SypI∆C-CFP, Syt-YFP have been previously described [START_REF] Pennuto | Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses[END_REF][START_REF] Pennuto | Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles[END_REF]. To generate the pVAMP2-EYFP vector the VAMP2 cDNA was amplified by PCR from the pECFP-VAMP2 vector [START_REF] Pennuto | Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses[END_REF] with the following oligonucleotides: forward, The expression plasmids for α-SNAP wild type and L294A mutant were a kind gift of Dr. R.D.

Burgoyne (University of Liverpool, Liverpool, UK).

Cell culture and transfections

Transformed human epithelial cells, HeLa cells, were grown on either plastic dishes or glass coverslips at 37°C in a 5% CO 2 humidified atmosphere in Dulbecco's modified Eagle's medium (Biowhittaker, Verviers, Belgium), supplemented with 10% fetal calf serum (Hyclone, Logan UT, USA), 1% L-glutamine and 100 U/ml penicillin/streptomycin (Biowhittaker).

For immunofluorescence studies, cells grown on glass coverslips were transfected with 1.5-3 µg of DNA using a standard Ca 2+ -phosphate precipitation protocol [START_REF] Kingston | Introduction of DNA into Mammalian Cells[END_REF]. Cells were analysed 48-72 hours later. For biochemical studies, cells grown on 6 cm plastic dishes were transfected with Lipofectamine2000 (Stratagene, La Jolla CA) according to the manufacturer's instructions, using 2-4 µg of each plasmid and 1.6 µl of Lipofectamine2000 per µg of plasmid. Transfected cells were analysed 24 hours later. In most of the experiments plasmids were co-transfected at equimolar ratio unless differently indicated. In order to widen the range of expression levels in the experiments shown in Fig. 4C, we pooled data from parallel samples co-transfected with plasmids in a ratio of 1:1 and 1:3.

Cell labeling protocols

Immunofluorescence experiments were carried out as previously described [START_REF] Menegon | Spatial and temporal regulation of Ca2+/calmodulindependent protein kinase II activity in developing neurons[END_REF]. For cell surface detection of fluorescent chimeras cells were incubated for [START_REF] Prekeris | Syntaxin 13 mediates cycling of plasma membrane proteins via tubulovesicular recycling endosomes[END_REF] applied twice for 2 minutes with a washing in cold KRH for 2 minutes in-between in order to remove surface-associated antibody prior to fixation and immunofluorescence analysis. In some cases, surface staining was carried out on fixed specimens, incubated with anti-GFP antibody in the absence of detergent and processed as described above.

For the trasferrin uptake assay, transfected cells were serum starved for 1 hour in the presence of BSA (2 mg/ml) prior to incubation for 30 minutes at 37°C in the presence of FITC-conjugated transferrin (30 µg/ml). After incubation, cells were subjected to acid-stripping at 4°C as described above to remove surface-associated transferrin prior to fixation.

Immunoprecipitation

Transfected cells were rinsed twice with cold phosphate-buffered saline (PBS), and harvested by scraping with PBS followed by centrifugation for 3 minutes at 1000 × g. Extraction was performed with 200 µl of extraction buffer (KCl 140 mM, EDTA 2 mM, Hepes-NaOH 10 mM (pH 7.4), 1%

(v/v) Triton X-100) supplemented with protease inhibitor cocktail (Sigma-Aldrich) for 1 hour at 4°C under rotation, followed by two sequential centrifugation steps for 5 minutes at 1000 × g and 10 minutes at 20,000 × g. GammaBind TM G-sepharose beads (Amersham Biosciences, Uppsala, Sweden) were washed in extraction buffer and incubated in the presence of 2.6 µg of anti-HA monoclonal antibody for 2 hours at 4°C under rotation. The beads were incubated overnight at 4°C with a volume of total cell extract containing 400 µg of proteins under rotation in the presence of protease inhibitors. The beads were recovered by centrifugation, washed in extraction buffer, resuspended in sample buffer, boiled and subjected to SDS-PAGE and immunoblotting as previously described [START_REF] Menegon | Spatial and temporal regulation of Ca2+/calmodulindependent protein kinase II activity in developing neurons[END_REF]. The supernatants from the immunoprecipitation procedure were analyzed in parallel.

Biotinylation assays

Cells were cooled at 4°C for 10 minutes in culture medium, rinsed twice with cold KRH and incubated on ice under rocking for 30 minutes with 0.75 mg/ml of cleavable EZ-Link Sulfo-NHS-SS-Biotin (Pierce Chemical).

Videomicroscopy and image analysis

For live imaging cells were incubated in KRH and maintained at either room temperature or 37°C. Specimens were viewed with a Zeiss (Oberkochen, Germany) Axiovert 135 inverted microscope equipped with epifluorescence optics. Images were recorded with a C4742-98

ORCA II cooled charge-coupled device camera (Hamamatsu Photonics, Hamamatsu City, Japan)

and processed using Image Pro Plus 4.5 (Media Cybernetics, Silver Spring, MD), Adobe Photoshop 6.0 (Adobe System, San Jose, CA) and the public domain program ImageJ (developed at the US National Institute of Health, Bethesda, Maryland). Prism (GraphPad Software, Inc., San Diego, CA) was used for statistical analysis. Acquisition parameters were maintained constant throughout any single experiment. For measuring colocalization, merged images were modified to the same background and three 6.4 µm 2 square-regions were selected for each cell.

Overlap coefficient was calculated for this set of images using a dedicated command of Image Pro Plus 4.5. The number of VAMP2-positive puncta was determined in the same set of images by performing granulometic filtering with the Gran filter plugin of ImageJ followed by automatic counting of particles selected with an appropriate threshold command which was maintained constant for all measurements. For measuring surface to total chimera localization, images were modified to the same background and the ratio between the intensity (number of pixels × average intensity) of anti-FP immunoreactivity to total intrinsic chimera fluorescence was determined in individual cells. Film densitometry was carried out using dedicated commands of ImageJ. The same background level was subtracted to all lanes. 9

RESULTS

VAMP2 is primarily targeted to the plasma membrane in non-neuronal cells

To establish an assay for the sorting determinants of VAMP2, a fluorescent chimera in which EYFP was fused to the intralumenal domain of VAMP2 (VAMP2-YFP) was expressed in HeLa cells that do not express neuron-specific molecules. The patterns of fluorescence of VAMP2-YFP and of a variant of ECFP bearing a farnesylation signal (CFP-F) which directs the protein to the plasma membrane were largely superimposable, indicating that exogenous VAMP2 was predominantly directed to the cell surface. Consistent with previous reports [START_REF] Feany | The synaptic vesicle proteins SV2, synaptotagmin and synaptophysin are sorted to separate cellular compartments in CHO fibroblasts[END_REF][START_REF] Jarousse | The AP2 binding site of synaptotagmin 1 is not an internalization signal but a regulator of endocytosis[END_REF], an analogous distribution was observed when EYFP-tagged synaptotagmin I (SytI-YFP) was co-expressed with CFP-F. In contrast, exogenous synaptophysin I fused to EYFP (SypI-YFP) was targeted to intracellular compartments and no major co-localization with CFP-F was visible (Fig. 1A).

Further evidence for prominent plasma membrane-localization of exogenous VAMP2 was obtained by surface staining of living VAMP2-YFP-expressing cells at 4°C with an antibody to the fluorescent epitope, which is exposed to the external milieu upon targeting of the chimera to the plasma membrane (Fig. 1B). The distribution of anti-FP immunoreactivity largely overlapped with that of intrinsic VAMP2-YFP fluorescence, indicating that the bulk of the exogenous protein was associated with the plasma membrane. Measurement of the immunoreactivity of surface-associated VAMP2-YFP relative to the total intrinsic fluorescence of the chimera in individual transfected cells revealed a dose-dependent linear increase in the fraction of exogenous VAMP2 targeted to the plasma membrane (Fig. 1C).

Consistent with its exclusive binding to the surface-localized chimera, the anti-FP antibody was effectively removed by acid-treatment of cells prior to fixation. Since fluorescence of GFP and its spectral variants is pH-sensitive [27, 28], acid-treatment also led to selective quenching of fluorescence of surface-associated VAMP2-YFP, thus allowing the exclusive detection of the chimera localized to intracellular compartments (Fig. 1B). We exploited this effect to provide a semi-quantitative measurement of the amount of exogenous VAMP2 present at the cell surface, Immunofluorescence analysis showed that intracellular VAMP2-YFP was associated with endosomes positive for both TfR and Rab5, likely reflecting its constitutive recycling route (Fig. 1D). In order to study endocytosis of exogenous VAMP2, VAMP2-YFP-expressing cells surface-labeled with the anti-FP antibody at 4°C, were subsequently incubated at 37°C for 20 minutes and processed for immunodetection of the internalized antibody after surface stripping of the residual antibody. Several endocytic vesicles containing VAMP2-YFP were observed, indicating that internalization of ectopically expressed VAMP2 was not prevented in this heterotypic context (Fig. 1E).

Synaptophysin I interacts with VAMP2 and controls its subcellular distribution

It has been reported that synaptophysin I controls the presynaptic targeting of VAMP2 in hippocampal neurons and this effect requires the formation of hetero-complexes between the two proteins [START_REF] Pennuto | Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles[END_REF]. We tested whether such sorting control could be reconstituted in a heterologous system by co-expression of VAMP2-YFP with ECFP-tagged synaptophysin I (SypI-CFP) in HeLa cells. Remarkably, a dramatic change in the distribution of exogenous VAMP2 was observed upon co-expression with synaptophysin I.

In the presence of SypI-CFP, VAMP2-YFP no longer accumulated at the plasma membrane but was selectively targeted to intracellular compartments where the two chimeras largely colocalized (overlap coefficient 0.97 ± 0.03, mean ± s.d.; n=40 cells) (Fig. 2A). This effect does not appear to be specific for HeLa cells, since it was also observed in transformed fibroblasts and epithelial cells of various origins, as well as in primary cultures of astrocytes derived from the rat cortex (data not shown). In contrast, exogenous synaptotagmin I (SytI-YFP) retained a prominent plasma membrane targeting even when it was co-expressed with SypI-CFP (Fig. 2A). At least a subset of organelles bearing both VAMP2-YFP and SypI-CFP contained also endogenous TfR (Fig. 2B). Synaptophysin I expression approximately doubled the number of VAMP2-YFPpositive puncta but did not vary the overlap between VAMP2-YFP and TfR (Fig. 2C).

The specificity of the effect of synaptophysin I on VAMP2 sorting may rely on the formation of VAMP2-SypI hetero-dimers. The formation of VAMP2-SypI complexes was detected biochemically in cells co-expressing VAMP2-YFP and HA-tagged synaptophysin I by immunoprecipitation with anti-HA antibody. Co-immunoprecipitation also revealed the formation of hetero-complexes between VAMP2 and a C-terminal truncated synaptophysin I which completely lacks the cytosolic tail of the protein (Fig. 2D).

Synaptophysin I selectively directs VAMP2 to intracellular compartments

To unequivocally determine whether synaptophysin I-mediated relocalization of VAMP2 impinges on its plasma membrane targeting, membrane proteins of transfected cells were biochemically isolated following a surface biotinylation assay. Plasma membrane proteins of cells expressing VAMP2-YFP and either synaptophysin I, C-terminal truncated synaptophysin I or the endosomal component Rab11 were incubated with biotin at 4°C and labeled proteins were recovered by streptavidin-Sepharose precipitation (Fig. 3A). Densitometry revealed that synaptophysin I expression led to a 70% reduction in the steady-state levels of exogenous VAMP2 at the plasma membrane (Fig. 3B). Importantly, the levels of surface-associated TfR were unaffected by synaptophysin I expression (Fig. 3C). Decrease in the surface levels of VAMP2 was not observed upon expression of either C-terminal truncated synaptophysin I or Rab11 (Fig. 3B). We chose Rab11 as a control for the effects of synaptophysin I on sorting since in non-neuronal cells both proteins localize to recycling endosomes and influence cholesterol levels in these compartments when expressed at high doses [START_REF] Holtta-Vuori | Modulation of cellular cholesterol transport and homeostasis by Rab11[END_REF] (data not shown).

We sought to study the effect of synaptophysin I on VAMP2 sorting at the single-cell level. To

4A,C).

To further substantiate the specificity of the effects of synaptophysin I on VAMP2 sorting we examined whether synaptophysin I could perturb the constitutive trafficking of syntaxin 13, a single-pass membrane protein which recycles between the plasma membrane and TfR-positive endosomal compartments [START_REF] Prekeris | Syntaxin 13 mediates cycling of plasma membrane proteins via tubulovesicular recycling endosomes[END_REF]. ECFP was fused to the intralumenal carboxy-terminal tail of syntaxin 13 and plasma membrane targeting of the resulting chimera (syntaxin13-CFP) was revealed by surface staining with anti-FP antibody upon co-expression with SypI-YFP (Fig. 4B,C). The ratio between surface-associated immunoreactivity to total intrinsic fluorescence of syntaxin13-CFP did not vary upon SypI-YFP expression (Fig. 4C). Thus, synaptophysin I selectively influences VAMP2 sorting without affecting the distribution of markers of the endosomal recycling pathway.

Synaptophysin I does not affect the rate and extent of VAMP2 endocytosis

The dose-dependent decrease in the surface levels of VAMP2 induced by synaptophysin I might be a consequence of enhanced VAMP2 endocytosis from the plasma membrane. To test this possibility cells co-expressing VAMP2-YFP and either soluble ECFP, used as a control, or SypI-CFP were surface labeled at 4°C with a cleavable biotin derivative (NHS-SS-biotin). Cells were warmed to 37°C for various periods of time to allow endocytosis of surface labeled proteins, and the label remaining at the plasmalemma was removed by reduction of the disulfide linkage prior to streptavidin-Sepharose precipitation (Fig. 5A,B). A significant amount of exogenous VAMP2 was internalized within 15 minutes of incubation at 37°C, becoming resistant to disulfide reduction. Remarkably, synaptophysin I did not influence the internalization rate of either VAMP2 or TfR precipitated from the same extracts.

Precipitation of biotin-labeled VAMP2-YFP and SypI-CFP from the same cell extracts allowed direct comparison of their internalization kinetics. VAMP2 and synaptophysin I showed a fairly similar rate of internalization during the first 15 minutes of chase, although the latter exhibited faster recycling to the plasma membrane in the next 30 minutes (Fig. 5C).

To obtain morphological evidence for the coupling between VAMP2 and synaptophysin I along the endocytic pathway, VAMP2-YFP was surface stained at 4°C with an anti-FP antibody in cells co-expressing SypI-CFP. Cells were chased at 37°C for 7 minutes to allow endocytosis, and processed for immunodetection of internalized VAMP2-YFP after removal of surface-associated antibody. SypI-CFP was detected in virtually all vesicles bearing endocytosed VAMP2-YFP, suggesting that internalization of the two proteins occurs through a common intermediate (Fig. 5D).

Recycling at the plasma membrane is dispensable for synaptophysin I-directed sorting of exogenous VAMP2

We sought to investigate whether synaptophysin I-mediated control of VAMP2 sorting occurred at the plasma membrane. Thus, we tested whether expression of dynamin bearing the K44A mutation, which inhibits endocytosis in a dominant negative manner [START_REF] Damke | Induction of mutant dynamin specifically blocks endocytic coated vesicle formation[END_REF], impaired the ability of synaptophysin I to recruit VAMP2 to intracellular compartments. Expression of dynamin K44A effectively abolished internalization of fluorescein-conjugated transferrin (Supplementary Figure 1). Cells expressing VAMP2-YFP, SypI-CFP and either wild-type dynamin or the K44A mutant were analyzed 3 days after transfection. VAMP2-YFP was mainly associated with SypI-CFPpositive intracellular compartments both in cells expressing wild-type dynamin and in those expressing the K44A mutant (the overlap coefficient for VAMP2-YFP versus SypI-CFP was 0.94 ± 0.05 and 0.96 ± 0.06 in the presence of wild-type or mutant dynamin, respectively; mean ± s.d.; n=20 cells per condition) (Fig. 6A).

The possibility that endocytic blockade could impair the formation of VAMP2/SypI heterocomplexes was assessed biochemically. VAMP2 was co-precipitated with synaptophysin I with equivalent efficiency in cells expressing either wild type or mutant dynamin (Fig. 6B).

As an alternative approach to test the contribution of trafficking at the plasma membrane in the synaptophysin I-regulated sorting of VAMP2, we exploited the dominant negative α-SNAP L294A mutant, whose expression inhibits fusion at the plasma membrane by preventing SNARE complex disassembly [START_REF] Barnard | Stimulation of NSF ATPase activity by alpha-SNAP is required for SNARE complex disassembly and exocytosis[END_REF]. Cells expressing VAMP2-YFP, SypI-CFP and either wild-type α-SNAP or the L294A mutant were analyzed 3 days after transfection. The recruitment of VAMP2-YFP to SypI-CFP-positive intracellular organelles was not impaired by α-SNAP L294A expression (the overlap coefficient for VAMP2-YFP versus SypI-CFP was 0.97 ± 0.04 and 0.95 ± 0.04 in the presence of wild-type or mutant α-SNAP, respectively; mean ± s.d.; n=23 cells per condition) (Fig. 6C). Similar results were obtained when cells were transfected with the α-SNAP constructs 36 hours prior to transfection of the VAMP2-YFP and SypI-CFP plasmids, in order to allow accumulation of the mutant protein (data not shown).

Collectively, these results indicate that trafficking at the plasma membrane is dispensable for both formation of VAMP2/synaptophysin I complexes and synaptophysin I-directed sorting of exogenous VAMP2.

DISCUSSION

This study shows that in a heterotypic context which lacks a specific pathway for SV biogenesis, synaptophysin I directs the sorting of VAMP2 to vesicles exhibiting low availability for constitutive exocytosis. In contrast, synaptophysin I does not influence the subcellular distribution of other membrane proteins, such as exogenous synaptotagmin I or markers of the endosomal recycling pathway. The evidence that synaptophysin I retains the ability to control VAMP2 sorting in the absence of a neuronal context emphasizes the intrinsic functional independence of the sorting determinants which direct the trafficking of SV proteins.

In keeping with previous studies reporting a differential subcellular localization for SV proteins heterologously expressed in non-neuronal cells [START_REF] Feany | The synaptic vesicle proteins SV2, synaptotagmin and synaptophysin are sorted to separate cellular compartments in CHO fibroblasts[END_REF][START_REF] Jarousse | The AP2 binding site of synaptotagmin 1 is not an internalization signal but a regulator of endocytosis[END_REF][START_REF] Johnston | Synaptophysin is targeted to similar microvesicles in CHO and PC12 cells[END_REF][START_REF] Cameron | Colocalization of synaptophysin with transferrin receptors: Implications for synaptic vesicle biogenesis[END_REF][START_REF] Linstedt | Synaptophysin is sorted from endocytotic markers in neuroendocrine PC12 cells but not transfected fibroblasts[END_REF], we have found that in HeLa cells synaptophysin I localizes to organelles partially overlapping with TfR-positive endosomes, while both synaptotagmin I and VAMP2 are primarily targeted to the plasma membrane.

It has been recently shown that in neurons during recycling SVs retrieve some important components, such as VAMP2 and synaptotagmin I, from large surface reservoirs whose size is determined by the rates of SV exocytosis and endocytosis [36][START_REF] Fernandez-Alfonso | Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling[END_REF][START_REF] Wienisch | Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical[END_REF]. Whereas the surface localization of synaptotagmin I in non-neuronal cells has been ascribed to the absence of a neuro-specific mechanism required for its internalization [START_REF] Jarousse | The AP2 binding site of synaptotagmin 1 is not an internalization signal but a regulator of endocytosis[END_REF][START_REF] Diril | Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling[END_REF], in the case of VAMP2 we show that the protein is readily endocytosed and recycled through TfR-/Rab5-positive endosomes. Therefore, the dose-dependent accumulation of VAMP2 at the plasma membrane in non-neuronal cells is likely to result from saturation of the capacity of the endocytotic machinery.

In neurons, VAMP2 accumulates at the plasma membrane when overexpressed in the absence of stoichiometric amounts of synaptophysin I, whereas the proportionate increase in synaptophysin I expression levels restores the exclusive localization of VAMP2 to synapses [START_REF] Pennuto | Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles[END_REF]. However, since the balance between exocytosis and endocytosis of membrane proteins determines their localization to specific organelles or subcellular compartments, it is essential to establish whether synaptophysin I governs the sorting of VAMP2 either by facilitating its targeting to SV precursors along the exocytic pathway or by favoring its recruitment from the plasma membrane.

The latter possibility is inconsistent with our endocytosis assays showing that the extent and kinetics of VAMP2 internalization are not influenced by synaptophysin I expression. In addition, a role for the plasma membrane as a major domain for VAMP2 sorting by synaptophysin I is not supported by the observation that expression of dynamin or α-SNAP mutants which block trafficking at the cell surface has no effects on the recruitment of VAMP2 to intracellular compartments. Nonetheless, VAMP2 and synaptophysin I are recycled at the cell surface perhaps through a common endocytic intermediate, consistent with previous observations in neurons [27] and PC12 cells [START_REF] De Wit | Synaptic vesicles form by budding from tubular extensions of sorting endosomes in PC12 cells[END_REF].

Thus, our results indicate that the essential step in order for synaptophysin I to direct VAMP2 sorting occurs along the exocytic pathway, at either the TGN or endosomal compartments, prior to delivery to the cell surface. The evidence that the plasma membrane is the first membrane acceptor at the level of which VAMP2 functions as a v-SNARE [START_REF] Martinez-Arca | A mutant impaired in SNARE complex dissociation identifies the plasma membrane as first target of synaptobrevin 2[END_REF] seems to suggest that synaptophysin I and VAMP2 are co-sorted at the level of the TGN into vesicles directed to the cell surface. Vesicles bearing synaptophysin I and VAMP2, probably assembled in heterocomplexes as documented for SVs [START_REF] Calakos | Vesicle-associated membrane protein and synaptophysin are associated on the synaptic vesicle[END_REF][START_REF] Washbourne | Vesicle-associated membrane protein-2 (synaptobrevin-2) forms a complex with synaptophysin[END_REF][START_REF] Edelmann | Synaptobrevin binding to synaptophysin: A potential mechanism for controlling the exocytotic fusion machine[END_REF][START_REF] Pennuto | Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses[END_REF], are likely to exhibit a reduced propensity to be consumed by constitutive exocytosis. Indeed, interaction with synaptophysin I limits VAMP2 availability for the formation of SNARE complexes [START_REF] Edelmann | Synaptobrevin binding to synaptophysin: A potential mechanism for controlling the exocytotic fusion machine[END_REF] and stimulation-induced release of VAMP2 from synaptophysin I appears to be a prerequisite for SV fusion [START_REF] Pennuto | Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses[END_REF][START_REF] Reisinger | The synaptophysin/synaptobrevin complex dissociates independently of neuroexocytosis[END_REF][START_REF] Bonanomi | Taipoxin induces synaptic vesicle exocytosis and disrupts the interaction of synaptophysin I with VAMP2[END_REF]. Consistently, the number of VAMP2-positive vesicles displays a 2-fold increase when synaptophysin I is present. In addition, since this effect is accompanied by a 70% decrease in the surface levels of VAMP2, it appears that synaptophysin I increases either the number of VAMP2 molecules accommodated onto each vesicle or the vesicle size. The latter possibility might be compatible with the documented interaction of synaptophysin I with cholesterol [START_REF] Thiele | Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles[END_REF], as well as with the hypothesized role of the protein in vesicle biogenesis [START_REF] Thiele | Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles[END_REF]. Indeed, when expressed at exceedingly high doses, synaptophysin I has the ability to selectively alter the morphology and recycling properties of TfR-positive endosomes, leading to accumulation of cholesterol in these organelles (our unpublished observations).

Protein-protein interaction is not sufficient in order for synaptophysin I to govern the sorting of VAMP2. Indeed, C-terminal truncated synaptophysin I still interacts with VAMP2, but is unable to determine its intracellular sorting. Thus, the long C-terminal cytoplasmic domain is not required for formation of either synaptophysin I homo-oligomers [START_REF] Pennuto | Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses[END_REF] or hetero-complexes with VAMP2, but appears to be important for the control of VAMP2 targeting to intracellular compartments. This domain is phosphorylated by Src and Ca 2+ /calmodulin-dependent protein kinase II [43-45] and contains binding sites for dynamin I [46] and the AP-1 adaptor protein γ-adaptin [START_REF] Horikawa | Interaction of synaptophysin with the AP-1 adaptor protein gamma-adaptin[END_REF], as well as an internalization signal required for constitutive endocytosis of synaptophysin I [START_REF] Linstedt | Endocytosis of the synaptic vesicle protein, synaptophysin, requires the COOH-terminal tail[END_REF]. Hence, the control of VAMP2 sorting by synaptophysin I might require both a direct interaction between the two proteins [START_REF] Pennuto | Synaptophysin I controls the targeting of VAMP2/synaptobrevin II to synaptic vesicles[END_REF] and additional modulatory processes, such as post-traslational modifications or recruitment of other factors to the complex, which are prevented by deletion of the cytoplasmic C-terminal domain.

The view of VAMP2 sorting by its negative regulator, synaptophysin I, at early stages along the secretory pathway has immediate corollaries bearing on the control of the delivery of SV proteins from the neuronal cell body to synapses mediated by VAMP2-positive axonal membrane carriers [49]. This model implicates synaptophysin I in escorting VAMP2 to the sites where exocytosis must take place exclusively after the arrival of the appropriate stimulus. This mechanism would ensure that SV precursors endowed with VAMP2 are kept unable to fuse until they reach their proper destination.

The reduced propensity for vesicle fusion imposed by synaptophysin I does not entirely account for the effect of the protein on VAMP2 sorting. Indeed, the selective control exerted by synaptophysin I on VAMP2 targeting implies that along the exocytic pathway synaptophysin I directs VAMP2 to vesicles distinct from those mediating surface delivery of the other membrane proteins tested, including exogenous synaptotagmin I and markers of the endosomal recycling pathway. These vesicles might be equivalent to the electron-translucent microvesicles containing exogenous synaptophysin I described previously in epithelial cells [START_REF] Leube | Topogenesis and sorting of synaptophysin: Synthesis of a synaptic vesicle protein from a gene transfected into nonneuroendocrine cells[END_REF]. These results argue for the coexistence in the same cell of a variety of programs for constitutive exocytosis which are subjected to specific control mechanisms and preferentially exploited by different classes of proteins for their delivery to the cell surface.

  . The PCR product was sequenced prior to digestion with XhoI/HindIII (restriction sites underlined) and cloned into the pEYFP-N1 plasmid (Clontech Laboratories, Inc., Palo Alto, CA). The syntaxin13-CFP expression vector was produced by cloning the syntaxin 13 cDNA (kindly provided by Dr. V. Faundez, Emory University, Atlanta, GA) in frame at the C-terminal of ECFP in the pECFP-N3 vector (Clontech). The pEGFP-Rab11 and pEGFP-Rab5 vectors were kind gifts of Drs. M. Zerial (Max Planck Institute, Dresden, Germany) and C. Bucci (Università degli Studi, Lecce, Italy), respectively. EGFP was substituted with ECFP by a NheI/BsrGI cut. The expression vector for farnesylated ECFP was generated from the pEGFP-F plasmid (Clontech) by substitution of EGFP with ECFP by a NheI/BsrGI cut. The expression plasmids for HA-tagged dynamin and dynamin K44A were a kind gift of Dr. S.L. Schmid (Scripp Research Institute, La Jolla, CA).

  Unreacted biotin was quenched by washing the cells three times during 10 minutes with ice-cold 50 mM Tris-HCl (pH 8) diluted in KRH. For determining surface expression, biotin-labeled cells were lysed as described above. For the endocytosis assay, biotinlabeled cells were incubated at 37°C for various times. Parallel samples left at 4°C to block 7 membrane trafficking were used as negative controls. After incubation, biotin remaining at the cell surface was removed by reducing the disulfide linkage with three 15-minute incubations with icecold sodium 2-mercaptoethanesulfonic acid (MESNA) cleavage buffer (100 mM MESNA, 50 mM Tris-HCl pH 8.6, 100 mM NaCl, 1 mM EDTA, 0.2% BSA) at 4°C. Residual MESNA was quenched by washing the cells three times during 10 minutes with ice-cold 120 mM iodoacetamide diluted in KRH before cell lysis. After centrifugation at 13,000 rpm for 10 minutes, biotinylated proteins were isolated by incubation of 70 µg of cell extracts with UltraLink immobilized streptavidin (Pierce Chemical) for 5-10 hours at 4°C under rotation in the presence of protease inhibitors. The beads were recovered by centrifugation, washed in extraction buffer, resuspended in sample buffer and subjected to SDS-PAGE and immunoblotting. Total cell extracts were analysed in parallel.

  based on quantification of YFP fluorescence intensity in VAMP2-YFP-expressing cells subjected to acid-treatment or left untreated before fixation. Quantification revealed that 84 ± 3% of VAMP2-YFP fluorescence derived from surface-targeted chimera (mean ± s.d.; n=70-90 cells per condition).

  this purpose, cells co-expressing SypI-CFP and VAMP2-YFP were surface stained with an anti-FP antibody to detect plasma membrane-associated VAMP2-YFP (Fig4A). The specificity of this staining was ensured by the fact that only surface-targeted VAMP2, but not synaptophysin I, exposes the fluorescent tag to the exterior of the cell. The ratio between surface-associated VAMP2-YFP immunoreactivity to total intrinsic fluorescence of the chimera was measured and correlated to SypI-CFP fluorescence in individual cells (Fig.4C). This analysis showed that the relative amount of plasma membrane-associated VAMP2-YFP was inversely related to the levels of expression of SypI-CFP.A similar analysis was carried out in cells co-expressing VAMP2-YFP and either ECFP-tagged C-terminal truncated synaptophysin I (SypI∆C-CFP) or ECFP-tagged Rab11 (CFP-Rab11). The subcellular localization of SypI∆C-CFP was similar to the distribution of full-length synaptophysin I, with a preferential targeting to intracellular compartments which also contained exogenous VAMP2. Likewise, exogenous VAMP2 was present in CFP-Rab11-positive 12 endosomes. However, neither SypΙ∆C-CFP nor CFP-Rab11 exhibited the ability to exert a dosedependent control of VAMP2-YFP localization observed with full-length synaptophysin I (Fig.

Figure 1 .

 1 Figure 1. Exogenous VAMP2 is predominantly associated with the plasma membrane.

Figure 2 .

 2 Figure 2. Synaptophysin I interacts with VAMP2 and leads to its redistribution to intracellular compartments. (A) Cells co-expressing SypI-CFP (green in the merge images) and either VAMP2-YFP (upper panel) or SytI-YFP (lower panel) (red in the merge images). Synaptophysin I directs VAMP2 to intracellular compartments, whereas it does not influence the plasma membrane localization of synaptotagmin I. (B) A subset of vesicles bearing both VAMP2-YFP and SypI-CFP contain also TfR (arrowheads). (C) Number of VAMP2-YFP-positive intracellular puncta and their colocalization with TfR measured in cells expressing VAMP2-YFP and either soluble ECFP (VAMP2-YFP, light grey bars) or SypI-CFP (VAMP2-YFP/SypI-CFP, dark grey bars) after quenching of surface EYFP fluorescence (mean ± s.d.). Synaptophysin I expression increases the amount of VAMP2-positive organelles (Student's t test, p < 0.0001, n=72 cells per condition), while it does not affect the extent of overlapping between VAMP2 and TfR (n=46 cells per condition). (D) Cells co-expressing VAMP2-YFP and either ECFP (VAMP2), HA-tagged SypI-

Biochemical Journal Immediate Publication. Published on 2 Mar 2007 as manuscript BJ20061907 © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

This work was supported by grants from the Italian Ministry of University (Cofin 2004 and2005 and FIRB to F.B. and F.V.) and from the Cariplo Foundation. The financial support of Telethon -Italy (grant n. GGP05134 to F.B and F.V.) is gratefully acknowledged.