Alejandro Sandoval

Norma Oviedo

Abir Tadmouri

Traudy Avila

Michel De Waard

Ricardo Felix
email: <rfelix@fisio.cinvestav.mx>

Oviedo Norma

Tadmouri Abir

Avila Traudy

De Waard Michel

Felix Ricardo

Campbell

Two PEST-like

Keywords: Blotting, Western, methods, Calcium, metabolism, Calcium Channels, chemistry, genetics, metabolism, Calcium Signaling, physiology, Calcium-Binding Proteins, antagonists & inhibitors, metabolism, Cell Line, Dipeptides, pharmacology, Dose-Response Relationship, Radiation, Electric Stimulation, methods, Humans, Immunoprecipitation, methods, Membrane Potentials, drug effects, physiology, radiation effects, Microfilament Proteins, antagonists & inhibitors, metabolism, Mutation, physiology, Patch-Clamp Techniques, methods, Protein Conformation, Protein Subunits, Recombinant Fusion Proteins, chemistry, genetics, metabolism, Time Factors, Transfection, methods Beta subunit, Ca channels 2+, MAGUK proteins, PEST sequences, calpain

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The major function of the voltage-gated Ca (Ca ) channels is to convert changes in membrane potential into an intracellular calcium 2+ V (Ca ) signal. Transient rises of Ca trigger or regulate diverse intracellular events, including metabolic processes, muscle contraction,

2+ i 2+ i
secretion of hormones and neurotransmitters, cell differentiation and gene expression. Several types of Ca channels have been V characterized and designated L, N, P/Q, R, and T. These channel types can be grouped into two major functional classes: high voltage-and low voltage-activated channels (HVA and LVA, respectively). The HVA Ca channel permeation pathway is formed by its subunit,

V α 1 which is encoded by a family of 7 genes ( ). The current through these channels may be modulated by distinct ). Site-directed mutagenesis of conserved serine residues in consensus sites for protein kinase phosphorylation suggests a role of 2004 phosphorylation in tuning the functional properties and pharmacological sensitivities of the Ca subunits ( ;

V β De Waard ., 1994 et al ;

). Gerhardstein ., 1999 et al Kohn ., 2003 et al These studies point to the importance of intramolecular domains in the physiology of the Ca subunits. In the current report, we show V β that Ca contains two PEST-like sequences (potential signals for rapid protein degradation), one in the SH3 domain and another in the V β 3 C-terminal end of the protein. These sequences are sensitive to low concentrations of calpain (a Ca -dependent protease). We in vitro 2+ further found that Ca mutants lacking the PEST sequences induced an increase in whole-cell Ca current amplitude compared to V β 3 2+ wild-type Ca , and caused a change in channel inactivation kinetic properties. Our findings suggest that Ca proteolytic cleavage may V β 3 V β 3 be an essential process for Ca channel functional regulation.

2+

Materials and Methods

Cell culture and recombinant Ca channel expression V Human embryonic kidney (HEK-293) cells were grown in DMEM-high glucose supplemented with 10 horse serum, 2 mM % L-glutamine, 110 mg/l sodium pyruvate and 50 g/ml gentamycin, at 37 C in a 5 CO /95 air humidified atmosphere. After splitting the μ °% 2 % cells on the previous day and seeding at ~60 confluency, cells were transfected using the Lipofectamine Plus reagent (Gibco BRL) with % 1.2 g plasmid cDNA encoding the rabbit brain N-type Ca channel Ca 2.2 pore-forming subunit (formerly ; GenBank accession

μ 2+ V α 1B
number D14157) ( ) in combination with 1.2 g cDNA coding the rat brain Ca -1 (M86621) ( ), and Fujita ., 1993 et al

μ V α 2 δ
Kim ., 1992 et al 1.2 g cDNA of the rat brain Ca (M88751) ( ) or its mutants (see below). For electrophysiology, 0.36 g of a

μ V β 3
Castellano ., 1993 et al μ plasmid cDNA encoding the green fluorescent protein (GFP; Green-Lantern; Gibco/BRL) was added to the transfection mixture to select positively transfected cells.

Deletions of Ca

PEST regions V β 3

The different deletions in the Ca subunit were obtained by the QuikChange XL-mutagenesis kit QCM (Stratagene), following a two V β 3 stage PCR protocol for deletions (Wang and Malcolm, 2001). The PEST regions 1 and 2 (amino acid residues 24 to 37 and 397 to 411, respectively) were subjected to deletion through duplex oligonucleotides that comprised the adjacent nucleotidic sequences to these regions. The forward oligonucleotides were 5 -GTTCAGCCGACTCTACACCAGAGAGTGCCCGGCGAGAAGTGG-3 and 5

′ ′ ′
-GAGGAGCATTCACCCCTGGAGCAGGCCTGGACCGGATCTTCACAG-3 for PEST1 and PEST2, respectively. Reverse ′ oligonucleotides were complementary to these sequences. In step I of the procedure, two extension reactions were performed in separate tubes, one containing the forward primer and the other including the reverse and complementary primer. Five polymerization cycles were conducted at 95 C 30 s, 55 C 1 min, and 68 C 14 min. After that, both reactions were mixed and the standard QCM procedure continued °°°f or 16 cycles. In addition to the single PEST deletions, a double deletion cDNA clone was obtained from the first plasmid harboring the PEST1 deletion in combination with the duplex oligonucleotides for the PEST2 deletion. Deletions were confirmed by either restriction endonuclease analysis (using endonucleases III and I) or automatic sequencing using an ABI PRISM 310 sequence analyzer Hind Xba (Perkin-Elmer Applied Biosystems) and primers for the T7 and SP6 promoters.

In vitro transcription and translation

transcription/translation assays were performed using the TNT Quick Coupled transcription/translation system kit In vitro ™ (Promega). Briefly, 2 g of plasmid DNA was added to 41 l of TNT quick master Mix containing 1 l of S -methionine (1000

μ μ ™ μ [ 35 ]
Ci/mmol) at 2.5 mCi/ml (Amersham Pharmacia Biotech) to a final volume of 50 l and incubated at 30 C for 120 min. Proteins were μ º subjected to SDS-PAGE (see below) and labeled proteins were detected by film exposure for 48 h.

SDS-PAGE and Western blotting

Microsomes from transfected HEK-293 cells were obtained as described elsewhere specific antibody was a sheep polyclonal antibody (1:1000 dilution; Sh0049), and the secondary antibody was a rabbit anti-sheep IgG horseradish peroxidase (Zymed) used at a dilution of 1:4000.

Pull-down experiments

As mentioned earlier, S -labelled proteins (wild-type , P1, P2 and P1 2) were expressed using a coupled

[ 35 ] β 3 β 3 Δ β 3 Δ β 3 Δ -
in vitro transcription/translation system as indicated by the manufacturer (TNT Kit, Promega). For pull-down assays, 75 l of hydrated ™ μ glutathione-agarose beads (Sigma) were incubated respectively with 20 g of GST fused to AID (Alpha1 Interaction Domain of Ca 2.2;

μ 2.2 v
) or of GST alone for 2 hours at 4 C. In order to saturate non specific free sites, GST-AID and GST beads were Sandoz , 2001 et al.

°2.2 incubated with 0.1 mg/ml BSA overnight at 4 C. S -labelled proteins were incubated with the beads for 1 h at room temperature. The ° [35 ] beads were then washed with PBS three times and the proteins, bound to beads, were eluted in denaturing buffer and analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by autoradiography.

Electrophysiology

Forty eight hours after transfection, cells expressing the GFP reporter gene were subjected to the whole-cell mode of the patch clamp

technique (

). In brief, Ba currents through Ca channels were recorded with an Axopatch 200B amplifier (Axon Hamill ., 1981 et al

2+ 2+
Instruments) and acquired on-line using a Digidata 1320A interface with pClamp8 software (Axon Instruments). After establishing the whole-cell mode, capacitive transients were canceled with the amplifier. Currents were obtained from a holding potential (HP) of 80 mV L-cysteine to each plate for 40 min at 37 C. To remove radioactive media, dishes were washed with PBS. Subsequently, normal DMEM °media supplemented with 10 FCS was added to plates (except the t 0 sample, which represents the start time of the chase). At t 24 h,

% = =
all plates were washed with ice-cold PBS. Cells were then scraped in 10 ml ice-cold PBS and transferred to a tube to remove the supernatant by centrifugation. Labeled cells were lysed with 1 ml PBS supplemented with 0.5 triton X-100 and a cocktail of protease % inhibitors (complete, Mini, EDTA-free, Roche). Lysates were sonicated and centrifuged at 1,500 rpm for 15 min. The supernatants were used for immunoprecipitation experiments. A total of 200 g proteins of each sample were incubated with anti-Ca polyclonal IgG for 1

μ V β 3
hr at room temperature. This polyclonal antibody was described elsewhere ( ), but was raised against the full-length Bichet , 2000 et al.

sequence of Ca . Subsequently, the Ca -IgG complex were immobilized by Protein A sepharose beads. Eluted proteins were then V β 3 V β 3 loaded on a 12 SDS-PAGE. After protein separation, the gel was treated for 30 min with a fixation solution (50 methanol, 10 acetic % % % acid) and 30 min with a solution of 10 glycerol, before a 24 hr autoradiography exposure.

%

Data analysis

The data are given as mean S.E.

Results

Although important progresses in the structure-function relationship of the Ca subunits have been made recently ( V β

Richards ., et al

), almost nothing is known about the mechanism and determinants of recognition for proteolytic degradation of these proteins. In this 2004 context, PEST domains are short sequences (10 60 residues) enriched in proline (P), glutamic/aspartic (E), serine (S) and threonine (T) residues, found in many short-lived eukaryotic proteins that play a role in their degradation (

). Using the Rechsteiner and Rogers, 1996

PESTFind algorithm (available at the URL https://emb1.bcc.univie.ac.at/toolbox/) we found one conserved PEST sequence (N-terminal; PEST1) and one variable (C-terminal; PEST2) PEST sequence in the Ca proteins. In the rat brain Ca subunit, the PEST1 region

V β V β 3
comprises amino acid residues 24 to 37, while PEST2 consists of amino acid residues 397 to 411 ( ). PEST1 scored 9.47 and Fig. 1A + PEST2 11.52, respectively. These findings prompted us to investigate the importance of the PEST sequences in the susceptibility of Ca

+ V β
to proteolytic degradation. It is worth mentioning that the Ca subunit is the major Ca constituent of the Ca 2.2 channel complex (

3 V β 3 V β V
), which plays a pivotal role in regulating neurotransmitter release and in controlling endocrine secretion. Scott , 1996 et al.

By using a two stages PCR protocol (see Methods section), we first created two single ( P1 and P2) and one double ( P1 2) PEST

Δ Δ Δ -
domain deletions in the Ca sequence. The plasmids containing the full-length sequence and the constructs harboring the deletions were V β 3 initially examined using a cell-free transcription/translation system. All the cDNA clones directed the synthesis of polypeptides of in vitro the expected molecular weights (

). Next, the wild-type Ca and its three PEST-deleted versions were expressed in HEK-293 cells Fig. 1B V β 3 and analyzed by immunoblotting using polyclonal antibodies directed against a fusion protein of the C-terminus of Ca ( V β 3 Liu ., 1996 et al ;

). This analysis revealed a single immunoreactive protein band with a molecular mass of ~58 kDa in microsomes from Scott ., 1996 et al transfected cells, which corresponds to the full-length Ca ( ). The antibodies also recognized the PEST-truncated proteins by V β 3 Fig. 1C showing shifts in the mobility from 58 kDa down to ~50 55 kDa on SDS-PAGE 10 gels (

). These findings, combined with the -% Fig. 1C lack of endogenous Ca subunit, make the HEK-293 cell line a good cell model to investigate the importance of the PEST sequences in V β 3 Ca function.

V β 3

We next questioned whether the PEST domains of Ca were molecular substrates for degradation by Ca -dependent

V β 3 in vitro 2+
endogenous proteases. This was tested through a comparative analysis of Ca -mediated proteolysis using the Ca mutant lacking both

2+ V β 3
PEST regions ( P1 2) and the full-length Ca subunit as substrates.

shows that recombinant Ca expressed in HEK-293

Δ - V β 3 Figure 2A
V β 3 cells was partially cleaved by endogenous proteases in the presence of CaCl . Though significant degradation was observed, proteolysis 2 was not complete; this could be explained by the fact that besides Ca no other agent was used to induce proteolysis. In addition,

2+

endogenous molecules that activate proteolytic activity (by reducing the Ca requirement, for instance), may be lacking in the cell 2+ homogenates employed in these assays. Hence, when Ca was present, the specific anti-Ca antibody detected additional bands that 2+ V β 3 should correspond to Ca fragments following the cleavage of the full-length Ca by Ca -dependent endogenous proteases. As

V β 3 V β 3 2+
expected, this proteolytic break-down of Ca was prevented by adding EDTA to the incubation buffer used for the experiments. Unlike

V β 3 the full-length Ca subunit, the P1 2 mutant was stable in the presence of Ca and did not undergo proteolysis in the

V β 3 Δ - 2+ EDTA-containing buffer ( ).
Likewise, examination of gels stained with Coomassie Blue provided initial evidence that a Fig. 2A ~58,000-Da polypeptide (presumably the Ca ) is susceptible to Ca /calpain-induced proteolysis. Hence, microsomes of HEK-293 cells

wild-type Ca

subunit is a target of this protease.

V β 3

We next sought to determine whether the PEST regions found in Ca were substrates of calpain. To this end, proteolysis V β 3 experiments of wild-type and mutant Ca proteins were conducted according to a protocol described elsewhere ( ).

V β 3 Shumway , 1999 et al.

Briefly, calpain activity was indirectly determined by assessing the extent of Ca -induced degradation of Ca P1 2 mutant subunits

2+ V β 3 Δ -
compared to the wild-type protein degradation ( ). Inhibitors such as EDTA and calpeptin were used in the assay to ascertain the Fig. 2C specificity of calpain activity. As described above, incubation with Ca (750 M) caused degradation of Ca which was prevented in

2+ μ V β 3
the presence of EDTA or calpeptin. On the other hand, examination of the Ca P1 2 mutant by Western blot evidenced that this protein

V β 3 Δ -
does not undergo endogenous Ca /calpain-induced proteolytic cleavage, illustrating the importance of the PEST sequences ( ).

2+

Fig. 2C As a more direct test of the contributing role of the PEST domains as calpain substrates, we examined the proteolytic profile of the Ca subunit mutants in which the PEST sequences had been removed. First, Ca was produced by translation in the rabbit

V β 3 V β 3 in vitro
reticulocyte lysate, then mixed with increasing concentrations of calpain in the presence of Ca to activate the protease. The results

2+

indicated that the translated Ca protein is indeed susceptible to exogenous calpain breakdown in a dose-and Ca -dependent in vitro V β 3 2+ manner. Notably, calpain concentrations of ~10 nM were sufficient to significantly degrade the translated Ca , and this effect in vitro V β 3 was prevented when EDTA (consistent with the Ca requirement of calpain) or the calpain inhibitor calpeptin were present. Of note, these 2+ experiments were performed adding exogenous calpain, in contrast to those in which the protein is degraded showing two additional bands (where proteolysis was mediated by endogenous Ca dependent proteases;

). We next questioned whether either the N-or the mutant proteins were almost completely degraded, proteolytic breakdown of these proteins seemed to be reduced in comparison to the full-length Ca at equal concentrations of calpain (not shown). Despite the fact that the internal concentration is unknown, we surmise V β 3 that a low concentration of the protease, in conjunction with the over-expression of our protein, explains that the effect is only partial. In contrast, when exogenous calpain is added, the proteolysis seems to be more potent. These results suggest that the PEST sequences within the Ca subunit promote its degradation by -calpain. In line with this, when the double PEST deletion mutant protein ( P1 2) was

V β 3 μ Δ -
incubated with calpain, we detected a significant decline in proteolysis relative to that of the single mutants. Following incubation with 50 nM calpain some of the input protein still remained ( ). Taken together, these data suggest that deletion of the PEST sequences Fig. 2D produced Ca subunits that were more resistant to cleavage by calpain than the full-length protein and provide evidence that the extent V β 3 of proteolysis depends on the presence of the PEST sequences.

In order to exclude the possibility that the reduced proteolytic sensitivity shown by the P1 2 mutant may be the result of gross Δstructural alterations in the Ca protein caused by the amino acids deletion, we next tested whether the PEST mutated versions of Ca

V β 3 Δ V β
could directly associate with the Ca subunit through the Alpha1 Interaction Domain (AID), the main channel structure involved in the 3 V α 1 Ca -Ca subunit interaction. To this end, we used a GST-fusion protein encoding a short fragment of the intracellular I-II loop of the

V α 1 V β
Ca 2.2 subunit carrying the AID region (GST-AID ), and compared its binding to the synthesized S -wild-type and mutant

V 2.2 in vitro [ 35 ] β 3 proteins ( ).
As can be seen, all of the S -Ca subunits maintained the ability to bind the GST-AID fusion protein indicating Fig. 3 [

35 ] V β 3 2.2
that the deletions in the PEST regions do not affect the association of these subunits to the purified GST fusion protein.

To extend these findings, we next investigated the functional repercussion of the PEST deletions in Ca by electrophysiological

V β 3 recording. The whole-cell mode of the patch clamp technique was used to study the macroscopic Ba currents ( ) through recombinant 2+ I Ba N-type Ca channels (composed of Ca 2.2 and -1) in HEK-293 cells transiently expressing wild-type Ca or its mutants.

V V α 2 δ V β 3 Figure 4A
shows representative current traces recorded during depolarizing voltage steps to 10 mV from a holding potential of 80 mV. Control

+ -
experiments carried out using cells transfected with the wild-type Ca showed that the average current density (peak current amplitude were also observed in cells expressing the single PEST deletions ( P1 and P2).

Δ Δ

The above described data are further illustrated in , which shows the density as a function of the voltage step in Figure 4C I Ba transfected cells. These current density-voltage relationships indicate that is activated at potentials positive to > 20 mV, and reach its I Ba peak at potentials close to 10 mV. The stimulatory effects of PEST deletions on current densities were observed at almost all potentials + explored. Interestingly, we noticed that PEST deletions also altered the macroscopic kinetic properties. Normalized currents obtained from either control or cells expressing Ca mutant subunits showed that the temporal course of the current traces was different ( ).

V β 3 Fig. 4A Though neither the time to peak nor the time constant for the activation of the current were apparently modified (data not shown), the time constant for the inactivation ( ) and the percentage of current remaining after 140 ms activating pulses were significantly different τ inact between control and cells expressing the mutant subunits ( ). It is worth mentioning that the use of Ba as the charge carrier in Fig. 4D 2+ these experiments may reduce Ca -dependent inactivation. Together, these results suggest that the PEST sequences may be important for 2+ determining also the effects of Ca subunits on channel voltage-dependent inactivation properties. Alternatively, it is also possible that V β kinetic modifications may be induced by PEST1 deletion through a non specific structural alterations of the Ca subunit. On the other V β 3 hand, the PEST2 region is less likely to be involved in such a non specific effect since previous studies have demonstrated that the third variable region (V3) of the protein (where PEST2 is located) may not be necessary for inactivation ( ; Wittemann ., 2000 et al Opatowsky et

).

., 2003 al

Given that the deletion of the PEST sequences in Ca resulted in mutant proteins more resistant to calpain cleavage ( ) that

V β 3 Fig. 2 enhanced functional expression levels of Ca channels ( ), we speculated that the calpain system may be responsible for the In contrast, longer exposure (6 h) to calpeptin resulted in a significant increase in density ( ). On the other hand, there was no I Ba Fig. 5B significant effect on density in HEK-293 cells expressing recombinant channels that included the Ca PEST mutant subunits after I Ba V β 3 calpeptin treatment (

), consistent with the idea that calpain proteolysis affects Ca activity through the presence of PEST Figs. 5C andD V β 3 sequences. Taken as a whole, the results presented above suggest that calpain may be a physiological regulator of Ca protein turnover.

V β 3

Lastly, as mentioned earlier, our data showed that the deletion of PEST like sequences decreases the degradation of Ca subunit V β 3 in ( ), we therefore aimed to reproduce these results . In order to determine whether the PEST region deletions affected vitro Fig. 2 in vivo protein stability, half-lives of the wild-type Ca subunit and its mutants were measured in metabolic pulse-chase experiments ( ). In V β 3 Fig. 6 these experiments, HEK-293 cells transiently transfected with the cDNA encoding the Ca subunits were labeled for 30 min with S

V β 3 [ 35 ]
-methionine/cysteine and chased with unlabeled methionine and cysteine. After 24 h of chase, proteins were immunoprecipited with a polyclonal anti-raised against the full-length Ca sequence to reveal most of the proteolytic fragments. As can be seen, the wild-type

β 3 V β 3
protein was synthesized as a band with a molecular mass of ~58 kDa which underwent progressive degradation (~85 was degraded after % 24 h). This result is consistent with the idea that proteins containing PEST sequences usually have short half lives, and is also consistent with our finding that Ca may be the target of proteolytic breakdown under physiological conditions. With the PEST mutants,

V β 3 Δ however, less degradation occurred during the 24 h of chase (~28, ~32 and 45 for P1 2, P1 and P2, respectively). This finding

% Δ -Δ Δ
suggests that the elevated surface expression of the channels containing the Ca mutant proteins may be related with enhanced protein V β 3 halflife.

Discussion

A number of proteins related to the metabolism or functions of intracellular Ca have been reported to be substrates for calpain intracellular Ca concentration. This led to the proposal that Ca channels could be degraded by a Ca -dependent protease. Interestingly,

2+ V 2+
Ca current rundown in myocytes has been shown to be accelerated by calpain and retarded by the physiological calpain inhibitor 2+ calpastatin, suggesting that these proteins may be involved in the regulation of channel activity and/or turnover ( ; Belles ., 1988 et al ).

Romanin ., 1991 et al

At first glance, the first PEST region which is highly conserved across species and Ca subunits appears of particular interest. The V β last few amino acids of this region contribute to an alpha helix that precedes the beta strands of the SH3 domain in several crystal structures of Ca ( ; ). Hence, if conserved sequence mediates conserved function, the V β 2a Opatowsky ., 2004 et al Van Petegem ., 2004 et al findings for Ca might be generalized to the entire family of Ca subunits. In addition, as we documented in the Results section, both

V β 3 V β
PEST regions showed modulatory effects on the functional expression of neuronal recombinant Ca channels.

V

In the present work, we show that calpain proteolysis may also affect the Ca auxiliary subunit, providing a novel mechanism for V β 3 modifying the regulation of Ca channels. As mentioned earlier, it has been suggested that calpain may cleave proteins near regions V containing PEST sequences ( ). It is proposed that these regions increase the local Ca concentration and, in Rechsteiner and Rogers, 1996 2+ turn, activate calpain. Analysis of the Ca sequence using the PEST-Find computer program revealed two PEST-like domains in the V β 3 protein ( ). Notably, though the presence of the PEST regions in the sequences of the Ca subunits and their possible roles in Fig. 1A V β subunit degradation was suggested initially several years ago ( ; ), their physiological relevance Ruth ., 1989 et al Perez-Reyes ., 1992 et al remain virtually unexplored. It is worth mentioning that proteins containing PEST sequences typically have short half lives (~2 h) in intact cells compared with most other proteins (>24 hours). In these proteins, removal or disruption of the PEST sequence increases the half life of the protein while insertion or creation of a new PEST sequence within a PEST sequence free protein decreases this half life.

Interestingly, it has been shown that the recombinant Ca subunit, when expressed alone in a mammalian cell line, is rapidly turned over

V β 3 (2 6 h) ( ).
In contrast to what we observed for the recombinant Ca used in this study ( ), which is consistent -Bogdanov ., 2000 et al V β 3 Fig. 6 with a rapid turnover of the protein, it has been noted that the half-life of native Ca subunits is about 50 h ( ). Though V β , 1995 et al. the reason for this discrepancy is unknown, it is possible that the association of the native Ca subunits with membrane bound proteins V β increases their stability.

Berrow

It is worth mentioning also that proteolytic cleavage within a PEST sequence may not serve for protein degradation only. In this regard, an exciting possibility is that proteolytical cleavage of the full length Ca may result in the generation of short forms of the V β 3 protein with potential physiological actions. This is particularly important after the identification of several novel fully functional short functional variants could be also formed by post-transcriptional processing of the protein.

On the other hand, the increase in Ca current amplitude induced by transfection of Ca mutant subunits lacking the PEST regions

2+ V β 3
in the HEK-293 cells suggest that the PEST mutants are more stable than the wild-type protein. In this scenario, the availability of Ca

Δ V β 3
would be enhanced which may reverse the inhibition imposed by the endoplasmic reticulum (ER) retention signal to the Ca subunit V α 1 facilitating the cell surface expression of the Ca channel complex. Indeed, PEST deletion seemed to make the PEST mutant proteins V Δ less susceptible to calpain cleavage ( ). Fig. 2 In addition, expression studies have shown that gating as well as regulation of high voltage-activated Ca channels is for a large part V determined by the interaction between the Ca and the Ca subunits. Though a range of functional effects has been identified for Ca ,

V α 1 V β V β
one of the most important actions of this protein is to facilitate the trafficking of the Ca subunit to the plasma membrane, partly by its V α 1 ability to mask the ER retention signal in Ca ( ). However, the Ca subunits can also affect the biophysical V α 1 Bichet ., 2000 et al ). Walker and De Waard, 1998 [START_REF] Arikkath | Auxiliary subunits: essential components of the voltage-gated calcium channel complex[END_REF]Campbell, 2003 Dolphin, 2003 In particular, diverse studies have revealed that Ca subunits have a marked effect on voltage-dependent inactivation of Ca ). Though the precise mechanisms of voltage-dependent inactivation are not well understood, Walker andDe Waard, 1998 Dolphin, 2003 it is clear that subunit composition differentially affects the inactivation properties of Ca channels. In general, wholecell patch clamp V studies indicate that co-expression of Ca , , and with Ca subunits do not modify the inactivation rate noticeably, whereas Ca

V β 1b β 2a β 4 V α 1 V β
markedly enhances inactivation ( ). Interestingly, our functional studies on the effects of the PEST sequences using Ca 3 Dolphin, 2003

V β 3 mutant subunits suggested a role for these regions in the regulation of neuronal N-type recombinant Ca channel activity by inactivation ( V ). Fig. 4D In response to membrane depolarization, control Ca channels quickly activate followed by rapid inactivation (

). In contrast, V Fig. 4A the channels containing mutant Ca subunits lacking the PEST regions displayed slower inactivation kinetics, resulting in a much V β 3 smaller fraction of inactivated channels at the end of the test pulse ( ). In each case, the halfactivation potentials closely aligned with Fig. 4D those observed in the presence of the wild-type Ca subunit, and therefore, any putative effects of the mutant subunits on inactivation V β 3 kinetics would unlikely be due to altered voltage dependence of activation gating. Interestingly, a variable region (V1) found at the N-terminal of Ca comprised of a short 14-amino acid stretch has been recently reported as a critical site for voltage-induced V β 3 inactivation ( ). In addition, a second variable region (V2) in combination with one of the conserved domains of the Stotz ., 2004 et al protein can also contribute to regulate Ca 2.2 inactivation (

). However, the PEST1 sequence in Ca is not located in a V Stotz ., 2004 et al V β 3 variable region of the protein, suggesting the presence of numerous domains in Ca capable of conferring rapid inactivation kinetics.

V β 3

Although diverse studies with Ca subunits suggest that some of their effects on channel activity require phosphorylation (

V β Dolphin,
) little is known regarding the role of this process on Ca channel inactivation. By using the NetPhos 2.0 software (available at the 2003 V URL

) which produces predictions for serine, threonine and tyrosine phosphorylation sites in http://www.cbs.dtu.dk/services/NetPhos/ eukaryotic proteins ( ), we found numerous phosphorylation sites for protein kinases on the Ca PEST-like sequences. Blom ., 1999 et al V β 3 Proteolysis of Ca by calpain would remove the phosphorylation consensus sites and the potential regulation of Ca channel inactivation V β 3 V through phosphorylation at these sites would also be abolished. Further studies are necessary to define whether the protein kinase-mediated regulation of the Ca subunit may be directly involved in the regulation of the inactivation process.

V β 3

The biochemical and electrophysiological studies described in the present study show that the Ca auxiliary subunit is sensitive to V β 3 μ -calpain digestion within its PEST-like regions and suggest that this enzyme may play a critical role in regulating Ca turnover. In V β 3 addition, the results provide evidence that the Ca PEST-like sequences might be regulatory segments that influence the V β 3 voltage-dependent inactivation properties of Ca channels. Lastly, if conserved sequence mediates conserved function, it would be V interesting to investigate whether the findings for Ca could be generalized to the entire family of Ca subunits.

Fig. 2

The Ca subunit is cleaved by Ca -dependent proteases. Recombinant Ca P1 2 protein is more stable than the wild-type Ca to

V β 3 2+ A, V β 3 Δ - V β 3
endogenous Ca -dependent proteases. and show the proteolytic breakdown of wild-type Ca ( ) heterologously expressed in radioactive media was replaced by complete medium; cells were lysed after a 24 h chase period in complete media, and analyzed by SDS-PAGE. An immunoprecipication with a polyclonal anti-IgG was performed to purify Ca variants and their proteolytic fragments.

β 3 V β 3
Lane 1 shows the control precipitation (incubation of protein A sepharose beads with protein extracts containing S -wild-type Ca ). Lanes

-

  and by applying test pulses every 20 s. Leak and residual capacitance currents were subtracted on-line by a P/4 protocol. Current signals were filtered at 2 kHz (internal 4 pole Bessel filter) and digitized at 5.71 kHz. Membrane capacitance (C ) was determined as described m previously () and used to normalize currents. The bath recording solution contained (in mM) 10 BaCl , 125 TEAglucose (pH 7.3). The internal solution consisted of (in mM) 110 CsCl, 5 MgCl , 10 EGTA, 10 HEPES, 4 Na-ATP and 0.1 2 GTP (pH 7.3). Experiments were performed at room temperature (~25 C).

  dishes with 40 confluent HEK-293 cells were transfected with cDNA encoding the wild-type Ca or its PEST% V β 3 Δmutants. 24 hours later, the protein labeling condition was set by incubating the cells for 30 min in methionine-and cysteine-free DMEM supplemented with 5 fetal calf serum (FCS; starvation period). Labeling was induced by adding 500 Ci S -L-methionine and 2 mM% μ [ 35 ]

  Figure 2Bof the microsomes with -calpain produced a dose-dependent change in the levels of the ~58 kDa protein in the gels. In contrast, the levels μ of the ~58 kDa polypeptide were unaffected when the Ca chelator EDTA or when the calpain inhibitor calpeptin were included in the 2+ assay. These data corroborate that the HEK-293 cell line possesses basal calpain activity ( ), and suggest that the Shimada , 2005 et al.

  Fig. 2A C-terminal PEST domains of Ca were molecular determinants for the efficiency of degradation by calpain. Recombinant Ca V β 3

  Figure 4B I Ba

  Fig. 4C regulated degradation of the Ca subunit . Hence, the observed increase in current density through Ca channels containing V β 3

2+including

  Ca channels. Initially, the demonstration that the Ca subunit was sensitive to calpain suggested a possible mechanism for V V α l regulation of Ca channel function. In the skeletal muscle, the C-terminal domain of the Ca 1.1 subunit is sensitive to proteolysis by proteolytic cleavage is thought to remove a major site of phosphorylation providing a mechanism for De Jongh ., 1994 et al modifying the cAMP-dependent regulation of L-type Ca channels. In addition, calpain proteolysis has been shown to affect the 2+ functional activity of Ca channels. In patch clamp studies, Ca currents decline progressively due to rundown which depends on the V 2+ " "

  channels by changing the rates of activation and deactivation by voltage as well as by altering the rate of voltage-induced V inactivation, the inhibition by G protein dimers, and/or the coupling of voltage sensing to pore opening ( ;

  key mechanism that contributes to the precise control of Ca entry into cells. Whilst the Ca subunit contains inherent 2+ V α 1 determinants of inactivation, association with different Ca subunits determines their overall inactivation rate ( ;

1 .

 1 Sandoval A , Felix R 2004; Intramembrane charge movement associated with endogenous K channel activity in HEK-293 cells + . Cell Mol Neurobiol. 24: 317-330 Ball SL , Powers PA , Shin HS , Morgans CW , Peachey NS , Gregg RG 2002; Role of the subunit of voltage-dependent calcium channels in the retinal outer plexiform β 2 layer. Invest Ophthalmol Vis Sci. 43: 1595-1603 Belles B , Hescheler J , Trautwein W , Blomgren K , Karlsson JO 1988; A possible physiological role of the Ca-dependent protease calpain and its inhibitor calpastatin on the Ca current in guinea pig myocytes. Pflugers Arch. 412: 554-556 Berrow NS , Campbell V , Fitzgerald EM , Brickley K , Dolphin AC 1995; Antisense depletion of -subunits modulates the biophysical and pharmacological properties of β neuronal calcium channels. J Physiol. 482: 481-491 Bichet D , Cornet V , Geib S , Carlier E , Volsen S , Hoshi T , Mori Y , De Waard M 2000; The I-II loop of the Ca channel subunit contains an endoplasmic reticulum Qin N , Olcese R , Tareilus E , Platano D , Costantin J , Stefani E 1998; Structures and functions of calcium channel subunits β . J Bioenerg Biomembr. 30: 357-375 Blom N , Gammeltoft S , Brunak S 1999; Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol. 294: 1351-1362 Bogdanov Y , Brice NL , Canti C , Page KM , Li M , Volsen SG , Dolphin AC 2000; Acidic motif responsible for plasma membrane association of the voltage-Jones JM , Meisler MH , Noebels JL 1997; Mutation of the Ca channel subunit gene Cchb4 is associated with ataxia and seizures in the lethargic (lh) mouse 2+ β . Cell. 88: 385-392 Castellano A , Wei X , Birnbaumer L , Perez-Reyes E 1993; J Biol Chem. 268: 3450-3455 Catterall WA 2000; Structure and regulation of voltage-gated Ca channels 2+ . Annu Rev Cell Dev Biol. 16: 521-555 Catterall WA , Striessnig J , Snutch TP , Perez-Reyes E 2003; International Union of Pharmacology. XL. Compendium of voltage-gated ion channels: calcium channels. Pharmacol Rev. 55: 579-581 Chen YH , Li MH , Zhang Y , He LL , Yamada Y , Fitzmaurice A , Shen Y , Zhang H , Tong L , Yang J 2004; Structural basis of the 1-subunit interaction of Foell JD , Balijepalli RC , Shah V , Hell JW , Kamp TJ 2005; Unique modulation of L-type Ca channels by short auxiliary subunit present in cardiac 2+ β 1d muscle. Am J Physiol Heart Circ Physiol. 288: H2363-H2374 De Jongh KS , Colvin AA , Wang KK , Catterall WA 1994; Differential proteolysis of the fulllength form of the L-type calcium channel subunit by calpain , Pragnell M , Campbell KP 1994; Ca channel regulation by a conserved beta subunit domain 2+ . Neuron. 13: 495-503 Dolphin AC 2003; subunits of voltage-gated calcium channels β . J Bioenerg Biomembr. 35: 599-620 Felix R , Gurnett CA , De Waard M , Campbell KP 1997; Dissection of functional domains of the voltage-dependent Ca channel 2 subunit 2+ α δ . J Neurosci. 15: 6884-6891 Foell JD , Balijepalli RC , Delisle BP , Yunker AM , Robia SL , Walker JW , McEnery MW , January CT , Kamp TJ 2004; Molecular heterogeneity of calcium channel beta-subunits in canine and human heart: evidence for differential subcellular localization. Physiol Genomics. 17: 183-200 Fujita Y , Mynlieff M , Dirksen RT , Kim MS , Niidome T , Nakai J , Friedrich T , Iwabe N , Miyata T , Furuichi T , Furutama D , Mikoshiba K , Mori Y , Beam KG 1993; Neuron. 10: 585-598 Gerhardstein BL , Puri TS , Chien AJ , Hosey MM 1999; Identification of the sites phosphorylated by cyclic AMP-dependent protein kinase on the subunit of L-type β 2 voltagedependent calcium channels. Biochemistry. 38: 10361-10370 Gregg RG , Messing A , Strube C , Beurg M , Moss R , Behan M , Sukhareva M , Haynes S , Powell JA , Coronado R , Powers PA 1996; Absence of the subunit ( ) of β cchb1 the skeletal muscle dihydropyridine receptor alters expression of the subunit and eliminates excitation-contraction coupling α Proc Natl Acad Sci USA. 93: 13961-13966 Gurnett CA , Felix R , Campbell KP 1997; Extracellular interaction of the voltage-Marty A , Neher E , Sakmann B , Sigworth FJ 1981; Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pfl gers Archiv ü . 391: 85-100 Hanlon MR , Berrow NS , Dolphin AC , Wallace BA 1999; Modelling of a voltage-dependent Ca channel subunit as a basis for understanding its functional properties 2+ β . FEBS Lett. 445: 366-370 Harry JB , Kobrinsky E , Abernethy DR , Soldatov NM 2004; New short splice variants of the human cardiac Ca subunit: redefining the major functional motifs V β 2 implemented in modulation of the Ca 1.2 channel V . J Biol Chem. 279: 46367-46372 Kim HL , Kim H , Lee P , King RG , Chin H 1992; Rat brain expresses an alternatively spliced form of the dihydropyridine-sensitive L-type Ca channel subunit 2+ α 2 . Proc Natl Acad Sci. 89: 3251-3255 Kohn AB , Roberts-Misterly JM , Anderson PA , Khan N , Greenberg RM 2003; Specific sites in the Beta Interaction Domain of a schistosome Ca channel subunit are key 2+ β to its role in sensitivity to the anti-schistosomal drug praziquantel. Parasitology. 127 : (Pt 4) 349-356 Laemmli UK 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685 Liu H , De Waard M , Scott VE , Gurnett CA , Lennon VA , Campbell KP 1996; Identification of three subunits of the high affinity omega-conotoxin MVIIC-sensitive Ca 2+ channel. J Biol Chem. 271: 13804-13810 McEnery MW , Copeland TD , Vance CL 1998; Altered expression and assembly of N-type calcium channel and subunits in epileptic lethargic (lh/lh) mouse Nunziato DA , Maltez JM , Prehoda KE , Pitt GS , Bredt DS 2004; Calcium channel function regulated by the SH3-GK module in beta subunits. Neuron. 42: 89-99 Olcese R , Qin N , Schneider T , Neely A , Wei X , Stefani E , Birnbaumer L 1994; The amino terminus of a calcium channel beta subunit sets rates of channel inactivation independently of the subunit s effect on activation ' . Neuron. 13: 1433-1438 Opatowsky Y , Chomsky-Hecht O , Kang MG , Campbell KP , Hirsch JA 2003; The voltagedependent calcium channel subunit contains two stable interacting domains β . J Biol Chem. 278: 52323-52332Opatowsky Y , Chen CC , Campbell KP , Hirsch JA 2004; Structural analysis of the voltagedependent calcium channel subunit functional core and its complex with the

  Fig. 1 Identification and deletion of the PEST-like sequences in the Ca subunit. , Schematic representation of the functional domains of Ca V β 3

  Fig. 3 Deletion of the PEST-like sequences did not alter the specific binding of the mutant Ca to the Ca subunit. Determination of Ca V β 3 V α 1 V β 3 binding to the AID of the Ca 2.2 subunit. Capacity of the wild-type and mutant S -Ca subunits to interact with the fusion protein V

Fig. 5

 5 Fig. 4 Deletion of the PEST-like sequences alters whole-cell in HEK-293 cells. , superimposed representative recordings obtained from I Ba A I Ba HEK-293 cells co-expressing neuronal recombinant Ca 2.2/ -1 channels and the wild-type Ca subunit (control) or its PEST deletion V α 2 δ V β 3 mutants ( P1, P2 and P1 2) in response to 140 ms test pulses to 10 mV from the holding potential of 80 mV. , Comparison of peak Δ Δ Δ -+ -

  5 represent the endogenous proteolytic degradation of the wild-type Ca subunit and its PEST mutations P1, P2 and P1 2V β 3 Δ Δ Δ -after immunoprecipitation by the anti-IgGs.

  β 3

  Detailed structural modeling of Ca subunits has proposed five discrete domains homologous to the membrane-associated guanylate V β kinase (MAGUK) protein family. Structural models have been proposed for the type 3 src-homology (SH3), and guanylate kinase

	phenotypes of mutant and knockout mice (	Gregg	et al	., 1996	;	Burgess	et al	., 1997	; McEnery	et al	., 1999;	Ball	et al	., 2002	). In-depth
	understanding of how the Ca auxiliary subunits modulate the activity of the ion-conducting Ca	subunits is essential for insights into
	(GK)-like domains (	Hanlon	et al	., 1999	;	Opatowsky	et al	., 2003	;	; 2004 McGee	et al	., 2004	;	Takahashi	et al	., 2004	;	Chen	et al	., 2004	;).
	SH3 and GK domains are both necessary to recapitulate full modulatory effects of Ca on the pore-forming Ca V β	V α 1	protein (	Takahashi et
	al	., 2004	;	McGee	et al	). Likewise, the C-terminus of Ca is associated to membrane targeting properties ( ., 2004 V β	Bogdanov	et al.	, 2000	)
	and the N-terminus appears to be involved in the regulation of channel inactivation (	Olcese	et al	., 1994	;	Restituito	et al	; ., 2000	Stotz	et al	.,
												Catterall	et al	., 2003
	structural modifications including the association with auxiliary subunits: the disulfide-linked Ca	, the intracellular Ca and the

V β

V α 1 the operation of HVA Ca channels in both normal and disease states.

V

  to the splice isoforms generated through the genetically encoded deletions of specific regions in the Ca gene, smaller

	variants of the Ca	V β 1	and Ca	V β 2	subunits (	Foell	et al	., 2004	;	Harry	et al	., 2004	;	Cohen	et al	., 2005	). In this context, our work might suggest
	that in addition

V β

  Sandoz G , Bichet D , Cornet V , Mori Y , Felix R , De Waard M 2001; Distinct properties and differential beta subunit regulation of two C-terminal isoforms of the P/Q-type De Waard M , Liu H , Gurnett CA , Venzke DP , Lennon VA , Campbell KP 1996; subunit heterogeneity in N-type Ca channels Mahon MJ , Greer PA , Segre GV 2005; The receptor for parathyroid hormone and parathyroid hormone-related peptide is hydrolyzed and its signaling properties are altered by directly binding the calpain small subunit. Endocrinology. 146: 2336-2344 Shumway SD , Maki M , Miyamoto S 1999; The PEST domain of I B is necessary and sufficient for in vitro degradation by -calpain Barr W , McRory JE , Chen L , Jarvis SE , Zamponi GW 2004; Several structural domains contribute to the regulation of N-type calcium channel inactivation by the beta 3 subunit. J Biol Takahashi SX , Miriyala J , Colecraft HM 2004; Membrane-associated guanylate kinase-like properties of -subunits required for modulation of voltage-dependent Ca

	Ca -channel 2+	α 1A	subunit . Eur J Neurosci. 14: 987-997
	Scott VE , β	2+	. J Biol Chem. 271: 3207-
	3212
	Shimada M , κ α	μ	. J Biol Chem. 274: 30874-30881
	Stotz SC , β	2+
	channels. Proc Natl Acad Sci USA. 101: 7193-7198
	Van Petegem F , Clark KA , Chatelain FC , Minor DL Jr 2004; Structure of a complex between a voltage-gated calcium channel -subunit and an -subunit domain
			Neuron. 42: 387-399
	Perez-Reyes E , Castellano A , Kim HS , Bertrand P , Baggstrom E , Lacerda AE , Wei XY , Birnbaumer L 1992; Cloning and expression of a cardiac/brain subunit of the β
	L-type calcium channel. J Biol Chem. 25: 1792-1797
	Rechsteiner M , Rogers SW 1996; PEST sequences and regulation by proteolysis. Trends Biochem Sci. 21: 267-271
	Restituito S , Cens T , Barrere C , Geib S , Galas S , De Waard M , Charnet P 2000; The	β 2a	subunit is a molecular groom for the Ca channel inactivation gate 2+ . J Neurosci.
	20: 9046-9052
	Richards MW , Butcher AJ , Dolphin AC 2004; Ca channel -subunits: structural insights AID our understanding 2+ β	. Trends Pharmacol Sci. 25: 626-632
	Romanin C , Gr sswagen

ö P , Schindler H 1991; Calpastatin and nucleotides stabilise cardiac calcium channel activity in excised patches. Pfiugers Arch. 418: 86-92 Ruth P , Rohrkasten A , Biel M , Bosse E , Regulla S , Meyer HE , Flockerzi V , Hofmann F 1989; Primary structure of the beta subunit of the DHP-sensitive calcium channel from skeletal muscle. Science. 245: 1115-1118 9 12

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Ackowledgements:

This work was supported by grants from Conacyt and The Miguel Aleman Foundation to RF. Doctoral (A.S.) and postdoctoral (N.O.) fellowships from Conacyt are gratefully acknowledged. We are in debt with Drs. B.A. Adams (Utah State University) and K.P. Campbell (University of Iowa) for their generous gift of the cDNA clones and antibodies. We thank G. Aguilar for assistance with DNA sequencing as well as A. Andrade and L. Escobedo for expert technical assistance.


Ability to use Galaxy interfaces

Various tables and graphical outputs are displayed to make the experience intuitive Workflow

Metabarcoding is the large-scale taxonomic identification of complex environmental samples via analysis of DNA reads of one marker gene (16S, ITS, 18S, COI, etc.).

The aim of metabarcoding analysis is to provide a table of abundance of OTUs/ASVs, as close as possible to the species, per sample as well as a descriptive statistical analysis of the composition of the targeted microbial population of the samples.

The various tools developed within FROGS [START_REF] Escudié | FROGS: Find, Rapidly, OTUs with Galaxy Solution[END_REF][START_REF] Bernard | FROGS: a powerful tool to analyse the diversity of fungi with special management of internal transcribed spacers[END_REF] offers such results. They allow users to process their data in command lines or via in a user-friendly Galaxy [START_REF] Afgan | The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update[END_REF] interface and to obtain different graphical and descriptive outputs.

FROGSFUNC: Smart integration of PICRUSt2 software into FROGS pipeline

Vincent DARBOT 1 , Moussa SAMB 1 , Maria BERNARD 2 , Olivier RUÉ [START_REF] Afgan | The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update[END_REF] Unlike metagenomics [START_REF] Thomas | Metagenomics -a guide from sampling to data analysis[END_REF] , metabarcoding does not provide these functional profiles of a microbial population, by being restricted to one marker gene.

PICRUSt2 [START_REF] Douglas | PICRUSt2 for prediction of metagenome functions[END_REF] bypass this restriction and obtain a prediction of the functional potential of a sample, at low cost.

Firstly, PICRUSt2 placed the marker gene (16S, ITS or 18S) sequences of interest into a its reference tree, that is used as the basis of functional predictions. After, it predicts number of marker and function copy number in each OTU. Then, for each sample, it calculates functions abundances and finally, pathway abundances are inferred, based on functional profile.

Functional inference: we assume that an organism has a metabolic function by the presence in the organism's genome of a known sequence having that function. FROGS highlights important information that are very useful for data interpretation. Some "hidden" PICRUSt2 outputs are exploited: reporting incongruence between taxonomic affiliations, Nearest Sequenced Taxon Index (NSTI) threshold confidence indicator, decision support graphic to help choosing the NSTI threshold

and Géraldine PASCAL 1 Background

 1 FROGSFUNC step1 placeseqs tool FROGSFUNC step2 copy numbers tool FROGSFUNC step3 functions tool FROGSFUNC step4 pathways tool PICRUSt2 placed the marker gene sequences (16S, ITS or 18S) of interest into a its reference tree, that is used as the basis of functional predictions Insert sequences Running hidden-state prediction to predict function abundances with castor-R of each OTUs placed in the PICRUSt2 reference phylogenetic tree Predicted gene copies number per sequence Prediction of functions weighted by the relative abundance of OTUs in the community. Function abundance predictions per sample Inferrance of MetaCyc/KEGG pathway abundances based on function number abundances. Pathway abundance predictions per sample 1 GenPhySE, Université de Toulouse, INRAE, ENVT, F-31326, Castanet Tolosan, France. 2 Univ. Paris-Saclay, INRAE, AgroParisTech, GABI, SIGENAE, F-78352, Jouy-en-Josas, France. 3 Univ. Paris-Saclay, INRAE, BioinfOmics, MIGALE bioinformatics facility, Jouyen-Josas, France Corresponding Author: vincent.darbot@inrae.fr

  Vincent Darbot's work has been supported by RESALAB OUEST http://frogs.toulouse.inra.fr

H 3 PO 4 reaction metabolic pathway What metabolic functions are presents in a microbial community ? Abundance table from FROGFUNC step1 Marker copy numbers table from FROGFUNC step2 Normalized OTU table by FROGFUNC step3 Normalized OTU table by FROGFUNC step3 Function copy numbers table from FROGFUNC step 2 Function abundances per sample by FROGFUNC step3

	one gene
	H 2 O
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