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(The FASEB Journal. 1999;13:1249-1257.)
© 1999 FASEB

A Sec7-related protein in Paramecium

SARASWATHY NAIR, CHARLES GUERRA and PETER SATIR1

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461-1602, USA

1Correspondence: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461-1602, USA. E-mail: satir{at}aecom.yu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have cloned and sequenced a SEC7-related gene in Paramecium tetraurelia that contains an open reading frame for 1135 amino acids encoding a 133 kDa protein, PSec7. Sec7, first identified in vesicular trafficking mutants in yeast, and its phylogenetic homologues function as guanine-nucleotide exchange factors for small G-proteins such as ARF (ADP-ribosylation factor). The deduced amino acid sequence in PSec7 for the motifs that form the ARF binding site are more than 70% identical to yeast Sec7 and similarly identical to ARNO, the human ARF exchange factor, with correct positioning of the critical glutamic acid residue within the motif region. Overall, the identity of PSec7 to yeast Sec7 is 32%. The deduced amino acid sequence also has five sequences that resemble IQ motifs, EF hand binding domains found in all myosins, and two pleckstrin homology domains. Similar sequences are present in yeast Sec7 and other Sec7-related molecules. A protein kinase A phosphorylation site may also be present. Southern blots suggest that a single gene encodes PSec7. Northern blots show that the message encoding PSec7 is induced on deciliation, followed by ciliogenesis, which suggests a role for PSec7 in cilia such as transport or targeting of ciliary membrane components.—Nair, S., Guerra, C., Satir, P. A Sec7-related protein in Paramecium.


Key Words: guanine nucleotide exchange factor • GEF • IQ motifs • cilia


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SEC7 WAS FIRST IDENTIFIED in Saccharomyces cerevisiae mutants in which there was accumulation of Golgi stacks and cisternae at the restrictive growth temperature of 37°C in low glucose medium (1 2 3) . The gene was isolated and found to encode a polypeptide of 2008 amino acids of ~230 kDa. The SEC7 gene product (Sec7) has been implicated in vesicular transport from various compartments of the Golgi apparatus (4) . Although Sec7 was found mainly in the cytoplasm, it could also associate with membrane fractions and was localized to the Golgi apparatus by indirect immunofluorescence (5) . Sec7-coated transport vesicles have been immunoisolated and visualized by electron microscopy (6) . It has been hypothesized that during vesicular transport, Sec7 is recruited from cytosol into the coat structure of non-clathrin-coated vesicles by protein–protein interactions.

In the past few years, several SEC7-related genes have been cloned in different species ranging from Arabidopsis thaliana and Caenorhabditis elegans to Homo sapiens (7 8 9) . In addition, at least three more SEC7-related genes have been identified from the Saccharomyces genome database. The Sec7-related proteins range in size from 43 kDa in humans to 163 kDa in Arabidopsis. Despite the differences in length, they all share a strong homology over an ~200 amino acid domain in the middle of the open reading frame, which is now called the `Sec7 domain'. In Arabidopsis mutants, cell division, elongation, and adhesion are defective due to a mutation in the EMB30 SEC7-related gene, where a highly conserved glutamic acid residue in the Sec7 domain is modified to a lysine residue (7) . This has implicated this residue as critical for Sec7 function.

A number of proteins containing Sec7 domains have been shown to possess guanine-nucleotide exchange factor (GEF)2 activity in that they promote replacement of GDP with GTP for fast activation of small G-proteins such as ARF (ADP-ribosylation factor). Among GEFs with Sec7 domains are ARNO (ARF nucleotide binding site opener), the GEF for human ARF-1, and several proteins closely related to ARNO (10 , 11) . ARNO also contains a pleckstrin homology (PH) domain near the carboxyl-terminal. Chardin and associates (12) have shown that the Sec7 domain of ARNO is itself a GEF that can stimulate nucleotide exchange efficiently. The 3-dimensional structure of ARNO has been described (13 , 14) . Sec7 domains feature two highly conserved sequences: in ARNO-Sec7 numbering, 151 FRLPGE 156 and 187 VL SFAV IMLNT SLH 200, which probably form the binding site for ARF (13 , 14) . ARFs are involved in recruiting coat complexes onto membranes during vesicular trafficking in mammalian cells (15) . Yeast Sec7 mutants are rescued by overexpressing human or yeast ARFs (16) . Sec7 probably plays a role in cycling ARF on and off membranes during normal vesicular trafficking. The Sec7 domain is also present on cytohesin, a cytoplasmic molecule that interacts with {alpha}1ß2 integrin (CD18) on Jurkat cells. Overexpression of either cytohesin or the Sec7 domain induces ß2 integrin-dependent binding of Jurkat cells to ICAM-1 (17) , which suggests that in this case Sec7 domain is important functionally in signal transduction events during intercellular adhesion.

We report here the cloning of a Sec7-related protein (PSec7) in the ciliated protozoan Paramecium. The deduced amino acid sequence of PSec7 incorporates an amino acid sequence derived from ciliary proteins of Paramecium. The Sec7 domain is highly conserved. We show that this protein has five sequences of truncated IQ motifs that are found in clusters along {alpha}-helices to either side of the conserved Sec7 domain, which suggests that EF hand molecules such as calmodulin can bind to this protein so that its function may be Ca2+ regulated. Similar sequences are present in related Sec7 proteins, including yeast Sec7. Secondary structure prediction suggests that two PH domains homologous to Yeast Sec7 PH domains are present in PSec7 near the carboxyl-terminal. Further, PSec7 mRNA is up-regulated on deciliation, followed by ciliogenesis, which suggests that the protein is involved with growth or function of cilia, possibly in trafficking, targeting, or operation of ciliary membrane proteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of genomic DNA
Methods were adapted from Subramanian et al. (18) . Two liters of 5-day, axenic cultures from Paramecium tetraurelia were harvested. The cell pellet was lysed by using a buffer containing 1% sodium dodecyl sulfate (SDS), 0.45 M EDTA, 0.1 M Tris-HCl pH 9.5, and 100 µg/ml proteinase K at 65°C for 30 min. The mixture was extracted with an equal volume of Tris-saturated phenol. The aqueous phase was collected and extracted with an equal volume of chloroform:isoamyl alcohol (24:1). The aqueous layer was collected; 3M sodium acetate was added to a final concentration of 0.3 M and the solution was layered carefully onto 2 volumes of cold ethanol into which the DNA precipitated. The DNA was spooled out using a glass rod. The sample was dissolved in 10 mM Tris, 1 mM EDTA, pH 8 (T.E. buffer), and dialyzed against the same buffer. The dialyzed sample was treated with DNase free RNase (1 mg/ml) for 1 h at 37°C. The sample was extracted again with phenol-chloroform, precipitated with ethanol, and dissolved in T.E. buffer.

Polymerase chain reaction
Polymerase chain reaction (PCR) was performed on genomic DNA isolated from Paramecium by using degenerate oligonucleotide sequences, biased for Paramecium codon usage, as primers. Primers were originally based on amino acid sequences of Paramecium ciliary proteins analyzed by high-performance liquid chromatography by Victor Freed (New York Medical College, Valhalla, N.Y.). PCR reaction components (~1 µg DNA, 10 mM Tris-HCl, pH 8.3, 50 mM KCl buffer, 1.5 mM MgCl2, 200 mM dNTPs, 50–200 µM primers) were mixed on ice and overlaid with mineral oil. The reaction components were warmed to 94°C for 3 min and held at 80°C. Then Taq polymerase (Perkin-Elmer, Branchburg, N.J.) was added and cycling was performed as follows: 94°C (30 s), 37–52°C (30 s), 72°C (1 min) repeated for 30 cycles, followed by 72°C (10 min). The PCR products were stored at 4°C (overnight). An aliquot of the reaction was analyzed on a 1% TAE gel.

Subcloning and sequencing
The PCR products were purified from the reaction mixture either directly or by gel purification with a Promega (Madison, Wis.) PCR purification kit. The purified product was subcloned by using a TA cloning kit (Invitrogen, Carlsbad, Calif., or Promega). The subclones were sequenced by using standard dideoxy procedures. Sequence analyses were originally performed with GCG (Madison, Wis.) sequence analysis software. The initial 750 base pair product, on which further cloning was based, contained an oligonucleotide sequence corresponding to the original ciliary protein sequence used to construct the primer at its 5' end and a 15 oligonucleotide match to the primer at its 3' end.

Isolation of mRNA
Three-day cultures of P. tetraurelia were deciliated with 5% ethanol following a modified Ogura and Machemer procedure (19) . The cells were allowed to recover for 2 or 3 h, then deciliated again and allowed to recover for 30 or 60 min. The cells were then harvested and mRNA was isolated following the manufacturer's protocol (Poly Attract system, Promega).

Reverse transcriptase-PCR
cDNA was synthesized by using oligo-dT or random primers and AMV reverse transcriptase (Invitrogen). Sense and antisense primers based on the sequence information from the genomic PCR product were synthesized. The PCR reactions were performed as above at annealing temperatures of 50°–60°C.

Northern blot
mRNAs from control (nondeciliated) and deciliated cells were resolved on 1.2% glyoxal agarose gels and transferred to Magnagraph nylon membranes (MSI, Westboro, Mass.) by capillary blotting in 20x SSC. The transferred RNAs were UV cross-linked to the blots (1200 joules/s). The blots were prehybridized with 5x SSC, 50% formamide, 1% SDS for 2 h at 42°C. The 750 bp DNA probe was gel purified and labeled with 32P-dCTP by random priming. The probe was added to fresh hybridization buffer and added to the blots for incubation at 42°C overnight. The blots were washed twice at room temperature for 15 min each with 2x SSC, 0.1% SDS, and then twice at 42°C for 15 min each with 0.5x SSC, 0.1% SDS. The blots were then exposed to film with an intensifying screen for appropriate lengths of time at -70°C. Controls included a chicken ß-tubulin DNA probe (kindly provided by Dr. D. Cleveland) and a probe from an unrelated Paramecium cDNA, labeled similarly.

Southern blot
Genomic DNA from Paramecium was digested with the restriction enzymes EcoRI, KpnI, BamHI, and HindIII at 37°C overnight. The restriction fragments were then resolved on a 0.8% TAE gel. The gel was denatured in 0.5 M NaOH, 1.5 M NaCl for 45 min and neutralized with 1.0 M Tris-HCl, 1.5 M NaCl. The gel was transferred to nylon membrane (Magnagraph, MSI) by capillary blotting in 20x SSC. The blot was further analyzed as described above or by using digoxigenin nonradioactive detection methods (Boehringer Mannheim, Indianapolis, Ind.).

5' and 3' RACE
For 3' RACE, sense gene-specific primers were synthesized. The cDNA was synthesized by using an oligo dT primer and reverse transcriptase in a reaction containing 50–500 ng of induced mRNA, 0.5 mM dNTPS, 2.5 mM MgCl2, 10 mM DTT, 20 mM Tris-HCl (pH 8.4), and 50 mM KCl buffer at 42°C for 1 h. PCR was performed with the cDNA as template and a gene-specific primer as the sense primer at 50°C annealing temperature, as described above. The PCR reaction was optimized with a second nested gene-specific primer.

For 5' RACE, gene-specific antisense primers were synthesized and used to construct cDNA from mRNA. The cDNA was dC-tailed by using terminal transferase (Life Technologies, Grand Island, N.Y.). PCR was performed using a polyG sense primer and a second nested gene-specific primer, as described above.

Secondary structure predictions and sequence comparisons
Secondary structure predictions for PSec7 were made using the methods of Guermeur et al. (20) or based on the crystal structure of ARNO (13 , 14) . Homology comparisons of amino acid substitutions and alignments in the Sec7 superfamily were made in part using POLINA (21) . Phylograms were generated by using GCG software with the Kimura algorithm for the distance matrix and the Neighbor-Joining algorithm to draw the tree (22) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of Paramecium Sec7-related protein
The SEC7-related gene was identified serendipitously in Paramecium during cloning of genes for ciliary proteins. Genomic DNA was used as template in PCR reactions using degenerate primers biased for Paramecium codon usage, based on partial amino acid sequences of ciliary proteins, which had previously been purified by salt extraction and SDS-polyacrylamide gel electrophoresis (23) . The initial PCR reaction from Paramecium genomic DNA generated several PCR products, one of which, a 750 bp product, contained an open reading frame coding for the deduced amino acid sequence (EQANQVDQ) at its 5' end. With a single nucleotide difference, this corresponded to the ciliary protein sequence L(T),E,Q,Q(D),N, Q,V,L(D),Q(E) used to construct the sense primer. When subcloned and sequenced, the deduced amino acid sequence from the 750 bp PCR product showed significant homology to yeast Sec7.

Paramecium genomic DNA was digested by restriction enzymes and probed in Southern blots with the 750 bp DNA probe (Fig. 1 ). A single 1 kb band was recognized after HindIII digestion and a single 2.5 kb band was seen after EcoRI digestion, which suggests that a single PSEC7 gene is present in the Paramecium genome, although additional blots will be needed to demonstrate this point conclusively. We used RACE cloning to isolate the entire cDNA encoding the Sec7-related protein. Figure 2 shows a schematic representation of the RACE cloning strategy and the lengths of the various PCR products generated using this strategy. We isolated cDNAs of ~2.0 kb using 3' RACE and ~1.5 kb, ~0.8 kb, and 0.15 kb using 5' RACE. Nucleotide sequence data was obtained from ~5000 bp of the RACE products, which is close to the full length of the message.



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  		Figure 1. Southern blot analysis of Paramecium DNA for PSEC7 genes. Genomic DNA from Paramecium (lanes 1–4) was digested with the restriction enzymes HindIII, KpnI, BamHI, and EcoRI, respectively. The digested DNA was resolved on a 1% TAE-agarose gel, transferred to a nylon membrane, and probed with a nonradioactively labeled 750 bp PSEC7 DNA probe (the original subcloned PCR product). On blots of up to ca. 3 kb in size under stringent conditions, single bands were recognized by the probe after HindIII or EcoRI digestion, as shown by the markers.



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  		Figure 2. Race and PCR cloning strategy to isolate the full-length PSEC7 clone. A schematic representation of the various PCR products obtained during the cloning of PSEC7. The shaded box shows the original 750 bp PCR product that was generated using primers from ciliary proteins. The various 3' and 5' RACE PCR products are shown by the lines with arrows. During the determination of sequence, some of the PCR products were generated from genomic DNA and had ~25–30 bp introns (typical of Paramecium) interrupting the ORF at the positions shown.

Figure 3 shows the nucleotide sequence and the deduced amino acid sequence of the full-length cDNA encoding the Paramecium SEC7-related gene. The original 750 bp probe, which begins at nucleotide 2348, is indicated. An open reading frame of 1135 amino acids is present, with a methionine at position 29 and a stop codon at position 3442 in the nucleotide sequence. The deduced amino acids encode a polypeptide of 133 kDa. The Sec7 domain extends from position 567 to 763 in the amino acid sequence. The two motifs responsible for ARF binding are highly conserved in PSec7 (motif 1, 83% identical to yeast Sec7; motif 2, 71% identical), and the glutamic acid residue that is critical for nucleotide exchange (13) is present in the correct position within motif 1 (Fig. 4 A). Structure prediction for PSec7 indicates that the proposed nucleotide exchange region (motif 1) lies on a short loop connecting two {alpha}-helices, as in ARNO, and that motif 2 forms part of another critical {alpha}-helix. Overall, the 1135 amino acid sequence of PSec7 shares 32% identity with amino acids 253 to 1550 of yeast Sec7.



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  		Figure 3. Sequence analysis of PSec7. The complete nucleotide and deduced 1135 amino acid sequence of the Sec7-related protein in Paramecium is shown. The untranslated 5' and 3' ends of the ~5.0 kb clone are ~28 bp and 1500 bp, respectively. The nucleotide sequence of the original 750 bp PCR product is shaded. The amino acid sequence derived from ciliary protein (aa 771–779) corresponding to the 5' primer is shown in boldface (not italicized). The nucleotide sequence corresponding to the 3' primer (3046–3060) is also indicated in boldface. The Sec7 domain (aa 567–763) is indicated in italicized bold letters, with motif 1 (aa 667–673) and motif 2 (aa 703–716) in black boxes. Truncated IQ motifs are indicated in shaded boxes. A putative PKA serine phosphorylation site (aa 284–288) is also shown in boxed italics.



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  		Figure 4. Amino acid conservation and secondary structure predictions for selected Sec7-related proteins. A) Comparison of amino acid conservation and structure in the Sec7 domain. Motif 1 forms a short loop between two helices. Motif 2 forms an {alpha}-helical domain. These regions are part of the binding site for ARF-1 in ARNO and are highly conserved in other Sec7-related proteins, including PSec7. Secondary structure based on ARNO crystal structure. B) Comparison of carboxyl-terminal regions containing PH domains in Sec7-related proteins. Boxed italics indicate the PH domains of PSec7. Two PH domains are present in tandem in each protein. Secondary structure based on Multivariate Linear Regression prediction (20) for PSec7. The amino- and carboxyl-terminal {alpha}-helices involved in the PH domains in PSec7 are part of highly conserved regions in other Sec7 proteins. Secondary structure notation: {alpha}-helices, slanted lines; ß sheets, arrows; random coils, solid lines (ARNO, based on crystal structure); dashed lines (PSec7, based on prediction); gaps in alignment are indicated by dots. Amino acid shading shows three or more identical residues in matched sequences using POLINA. Bold letters indicate positions of truncated IQ motifs in one or more sequences.

The Sec7 domain in PSec7 is flanked by truncated IQ motifs (Fig. 3 , gray boxes). The full-length IQ motif `IQXXXRGXXXR', where calmodulin or light chain binding occurs in myosins (24) , is shortened to I/L/VQXXXR/K. Five such motifs are present in PSec7, arranged in clusters. Similar motifs are present in yeast Sec7 and other Sec7-related proteins (Table 1 ), often in corresponding positions (Table 2 ).


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  		Table 1. List of IQ motifs in selected myosins and Sec7-related proteinsa


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  		Table 2. Matching IQ motifs in Sec7-related proteinsa

Sequence homology predictions indicate that immediately carboxyl-terminal to the Sec7 domain in PSec7 is a PH domain, homologous to similar domains in yeast Sec7 and ARF-GEP1 (Fig. 4B ). As in pleckstrin (25) , yeast Sec7, and ARF-GEP1, following this domain (after a spacer), a second PH domain is found in PSec7. The first PH domain of PSec7 runs from amino acids 780 to 921; it contains the final truncated IQ motif at its carboxyl terminus. The second PH domain of PSec7 runs from amino acid 1010 to 1125. Amino acid conservation of ARF-GEP1, yeast Sec7, and PSec7 (amino acids 960–1124) is particularly evident (Fig. 4B ). Part of this region corresponds to the 3' end of the original cloned 750 bp product. There is also a putative protein kinase A (PKA) histone-1-like serine phosphorylation site (RRQAS) at amino acids 284–288 in PSec7.

PSec7 mRNA is up-regulated during ciliogenesis
When ciliated cells are deciliated and allowed to regenerate cilia, the mRNAs for ciliary proteins are up-regulated whereas other cellular mRNAs generally remain unaffected (26 , 19) . Figure 5 shows a Northern blot of mRNA from nondeciliated cells vs. deciliated cells, which were allowed to regenerate for 2 or 3 h after a first deciliation, and 30 or 60 min after a second deciliation. The extra hour of regeneration before the second deciliation makes little difference in the amplification response. After 30 min recovery from the second deciliation, the mRNA for tubulin is amplified, as expected, by x5.4. After 60 min recovery, the tubulin mRNA is amplified further (x7.2). The blot probed with a 750 bp DNA probe from the PSEC7-related gene shows amplification (x3.9) of a 5 kb message after 30 min recovery from the second deciliation; after 60 min, recovery amplification is unchanged (x3.5). The blot probed with an unrelated probe from a nonciliary protein shows constant levels of message, confirming that induction is specific for ciliary proteins. This supports the suggestion based on amino acid sequencing of the original ciliary extract that PSec7 is located in the cilia or is transported there, perhaps during ciliogenesis.



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  		Figure 5. Induction of PSEC7 message on deciliation. mRNA was isolated from nondeciliated cells (lane 1) and deciliated cells, which were allowed to recover for 2 (lanes 2, 3) or 3 h (lanes 4, 5). After a second deciliation, cells were allowed to recover for 30 or 60 min, as indicated, and mRNA was isolated. After resolving the mRNA on a 1.2% glyoxal-agarose gel, the gel was transferred to nylon, probed with various radioactively labeled probes, developed, and quantitated using a scanning densitometer. Lanes 2–5 are compared to lane 1 to show whether induction has occurred. The top panel is a tubulin control. The bottom panel has been probed with an unrelated clone. The middle panel probed with the PSec7. 750 bp probe shows a x3.9 induction of message by 30 min after the final deciliation. Unlike tubulin, the induced message shows little increase as recovery proceeds for longer time. The sizes of the various induced messages are indicated.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have fortuitously cloned a Sec7-related protein in Paramecium in the process of PCR cloning other ciliary genes. An open reading frame from the ~5 kb cDNA, isolated by using RACE techniques, encodes a polypeptide with a deduced molecular mass of 133 kDa containing canonical Sec7 motifs within a ca. 200 amino acid Sec7 domain in the middle of the molecule, followed by two carboxyl-terminal pleckstrin homology domains. The Sec7 domain present in ARNO, a human guanine-nucleotide exchange protein whose 3-dimensional crystal structure has been solved, promotes guanine-nucleotide exchange on ARF1, an important molecule involved in vesicular coat formation (12 13 14) . The highly conserved motifs 1 and 2 of ARNO, including the invariant glutamic acid as the carboxyl-terminal residue of motif 1, are present in all Sec7-related proteins, including PSec7. Structure predictions for PSec7 in this region (compared with ARNO) indicate that amino acids 677–682 containing motif 1 (FTLPGE) lie between two highly conserved {alpha}-helical domains of the molecule, and that the relative structures and positions of the {alpha}-helix formed by amino acids 714–727 containing motif 2 (TLSYLLMMLQTDLH) and the PH domains that immediately follow are also conserved. These considerations support the prediction that PSec7 will also function as a guanine nucleotide exchange factor in conjunction with an ARF-like G-protein. However, Fig. 6 constructs a phylogenetic tree for a variety of Sec7-related proteins, including PSec7, showing that PSec7 and ARNO are only distantly related evolutionarily. The cytoplasmic or membrane localization, the G-proteins involved, and the physiological signal transduction pathways in which PSec7 functions are likely to be different from those of the ARNO-ARF1 or cytohesin pathways, since PSec7 probably plays some role in ciliogenesis or ciliary function, possibly in the targeting, assembly, or operation of ciliary membrane proteins.



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  		Figure 6. Phylogram of selected Sec7-related proteins. In constructing this tree, the Sec7 domain flanked by IQ motifs (PSec7 aa 378–918) was used to generate sequence comparisons. PSec7 is most closely related to yeast Sec7 and ARF-GEP1.

PSec7 contains five truncated IQ motifs, `IQXXXR', `LQXXXR/K', or `VQXXXK'; two clustered on one side of the Sec7 domain, three on the other. Truncated IQ motifs flanking the Sec7 domain are a feature of yeast Sec7 and most other Sec7 homologues, including ARNO, which has gone without previous comment. In yeast Sec7, the truncated IQ motif `LQLISKG' occurs in the same relative position as `LQLISRL' in PSec7, and a similar match is found between other Sec7 proteins (Table 2) . Truncated IQ motifs often occur in {alpha}-helical regions of the molecules and in clusters. In yeast Yec2, the motifs `LQLYPRL' and `IQNTTKL' occur as a cluster, whereas the cluster `LQHKPKL' and `IQLIWRV' occurs in a similar position in PSec7. Full-length IQ motifs, IQXXRGXXXR, known to bind EF hand proteins, are not found; however, it is assumed that the truncated IQ motifs perform a similar function. In myosins, both full-length and truncated IQ motifs are found in tandem repeats (23 ; Table 1 ). This raises the question of whether in Sec7 and related proteins (including PSec7), these motifs are important in binding EF hand proteins, such as calmodulin or centrin, in a physiologically significant manner. Both calmodulin and centrin are present in crucial locations in protistan cilia. Calmodulin is found both in conjunction with Ca2+ pumps at the ciliary membrane (27) and along the axoneme (28) . Centrin is also present along the axoneme bound to certain inner arm dynein heavy chains (29) , but more specifically it is part of the apparatus involved in severing cilia from the cell body in the transition zone (30) , where there are fibers connecting axoneme and membrane. Calmodulin and centrin are the putative Ca2+ sensors for behavioral and morphogenetic responses of cilia, including ciliary reversal in Paramecium. It is also possible that the putative PKA phosphorylation site in PSec7 is physiologically significant, as cAMP also plays a signal transduction role in Paramecium cilia (31) .

PH domains, a structurally conserved element found in many signaling proteins (32) , are present in proteins such as dynamin and Sec7, which are functionally interactive with membranes. It is noteworthy that a truncated IQ motif forms the carboxyl terminus of the first PH domain in both PSec7 and yeast Sec7, and that a truncated IQ motif forms the carboxyl terminus of the second PH domain in yeast Sec7 and human pleckstrin (Table 2) . This suggests that PH domains in these proteins may have a specific role in Ca2+ signaling.

At present, localization of PSec7 to cilia is based on 1) The presence of the original peptide derived from ciliary extracts, used in construction of the initial 5' sense primer for PCR, which was found in the amino acid sequence of PSec7, and 2) the increase in mRNA encoding PSec7 on deciliation, which was seen in Northern blots. This localization, if confirmed, would suggest that the PSec7 is involved in transport or targeting of ciliary components during ciliary generation or as an axonemal or ciliary membrane-matrix protein in ciliary function. Because of the presence of IQ repeats and PH domains, it is tempting to speculate that PSec7 is a functional component of the membrane skeleton of the ciliary membrane or its precursors that responds to changes in Ca2+ concentration. Sec7-related proteins are evolutionarily widespread. Figure 6 shows that in the sequences listed, PSec7 is on a separate branch closely related to Yeast Sec7. Yeasts, of course, do not possess cilia (or eukaryotic flagella), but ciliary growth and function are critical features of many organisms, including humans. Therefore, PSec7 is a recognizable Sec7, probably with a novel, physiologically significant cellular location whose orthologs may exist in many ciliated cells. The role of a PSec7-type guanine-nucleotide exchange factor in the building or function of the cilium in metazoan organisms is an intriguing question posed by these results and is probably not unique to PSec7.


   ACKNOWLEDGMENTS
 
We thank Sergei Levin for help with sequence alignment and secondary structure prediction. Toshikazu Hamasaki and Kurt Barkalow originally isolated the ciliary proteins used for microsequencing. We thank Victor Freed for microsequencing. Birgit H. Satir, Mitchell Bernstein, and Andrew Eisen provided help with molecular biology methodology, and Andrew Popper helped with cloning. This work was supported in part by grants from the USPHS (RR09875 and DK41918) and The American Heart Association, New York City Affiliate. S.N. was supported by a postdoctoral traineeship on DK07218.


   FOOTNOTES
 
2 Abbreviations: ARF, ADP-ribosylation factor; GEF, guanine-nucleotide exchange factor; ARNO, ARF nucleotide binding site opener; PCR, polymerase chain reaction; PKA, protein kinase A; PH, pleckstrin homology; PSec7, Sec7-related protein; SDS, sodium dodecyl sulfate.

Received for publication November 19, 1998. Revision received February 5, 1999.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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