Keywords: b/c-crystallin domain; Cysteine-rich region; Signal transduction
1. Introduction mone and neurotransmitter secretion, is also dependentupon increases in intracelluar Ca2+ concentration
Ca2+ is an important second messenger in many (Clapham, 1995).biological systems, regulating a wide variety of physio- Analogous to observations made in other systems,logical processes including cell proliferation, organiza- Ca2+ signaling is important for cellular functions intion of the cytoskeleton, cell motility, and modulation Paramecium. For instance, a cytosolic Ca2+ wave isof other second-messenger systems (Crivici and Ikura, believed to lead an elaborate scheme of duplicating and1995; Clapham, 1995). For example, in many cell types, re-organizing the pattern of ciliary basal bodies andtransient increases in the intracellular concentration of their associated cytoskeletal structures and networksCa2+ are closely correlated with entry into mitosis. during each cell division, such that its characteristicStimulus-dependent exocytosis, which is crucial for hor- asymmetry and polarity is maintained (Jerka-Dziadosz
et al., 1992). Ca2+ is also the main charge carrier ofAbbreviations: BAA, basic amphiphilic alpha; CaM, calmodulin; the Ca2+ action potential of Paramecium and, therefore,
EGF, epidermal growth factor; OG, n-octyl-b--glucopyranoside; has a major role in controlling membrane excitabilityORF, open reading frame; PCM, Paramecium calmodulin-binding and consequently swimming behavior (Schultz et al.,membrane-bound protein; PCR, polymerase chain reaction; SDS–
1990). The discharge of trichocysts (secretory organ-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.* Corresponding author. Present address: 1525 Linden Drive, elles), a process similar to regulated secretion in other
Madison, WI 53706, USA. Tel. : +1 (608) 262-7976, +1 (608) organisms, is also regulated by Ca2+ in Paramecium262-9472; Fax: +1 (608) 262-4570 ( Kerboeuf et al., 1993).
E-mail address: [email protected] (Ching Kung)Ca2+ acts through Ca2+-binding proteins, the most1 Present address: Biotechnology Center, University of Wisconsin-
Madison, 425 Henry Mall, Madison, WI 53706, USA. ubiquitous of which is calmodulin (CaM). CaM is a
highly conserved protein. Paramecium CaM is 88% -aspartate receptor (Zhang et al., 1998), and the small-conductance Ca2+-activated K+ channel ( Xia et al.,identical and 94% similar to bovine CaM ( Kung et al.,
1992). The crystal structure of native or heterologously 1998).We do not wish to further study known Ca2+/CaMexpressed Paramecium CaM is also nearly identical to
that of bovine CaM (Ling et al., 1994). It typically has targets and, therefore, did not try to clone theirhomologs in Paramecium by sequence homology. Wefour functional Ca2+-binding pockets of the EF-hand
type (Crivici and Ikura, 1995). Upon Ca2+-binding, instead directed our research towards finding newCa2+/CaM-binding proteins in the ciliary membrane.CaM changes its conformation, thereby enabling its
interaction with target proteins. This interaction, in This membrane is interesting because it containsimportant components of signal transduction, such asturn, induces conformational changes in the target pro-
teins and results in their altered activities (Crivici and depolarization-activated Ca2+ channels, guanylyl andadenylyl cyclases, type I protein phosphatases andIkura, 1995).
The roles of CaM in various Ca2+-regulated processes Ca2+-ATPases (Schultz et al., 1990). Here, we reportthat we have identified and purified a Ca2+-dependentin eukaryotes are still under investigation (e.g., see
Zhang et al., 1998 and Xia et al., 1998). However, CaM-binding protein, PCM1 (Paramecium CaM-bind-ing membrane-bound protein), from the ciliary mem-Paramecium is one of the few experimental systems
where viable CaM mutants have been found ( Kung brane of Paramecium. A corresponding partial cDNAclone for PCM1 has been obtained, and we furtheret al., 1992; Ling et al., 1994), and analyses of these
CaM mutants support the notion that CaM mediates discovered a family of PCM1 homologs, suggesting thatthe PCM family consists of at least four members.Ca2+ signaling in many cellular events. Phenotypes of
Paramecium CaM mutants suggest that CaM regulates Predicted amino-acid sequences are consistent with theidea that the PCM proteins are novel, with b/c-crystallinion channel functions, particularly those controlling
membrane excitation ( Kung et al., 1992; Ling et al., domains and cysteine-rich regions that confer structurespossibly important for their function(s).1994). Moreover, regulation of one of these channels,
the Ca2+-dependent Na+ channel, has been indicatedto be through direct binding of CaM (Saimi and Kung,1994). These CaM mutants also show pleiotropic defects 2. Materials and methodsthat are consistent with a role of CaM in regulatingmotility, stimulus-dependent exocytosis and growth and 2.1. Obtaining ciliary membrane proteins of
P. tetraureliadevelopment ( Kung et al., 1992; Ling et al., 1994;Kerboeuf et al., 1993). Biochemical and pharmacologi-cal studies in Paramecium offer additional collaborative P. tetraurelia strain nd-6 (Ling et al., 1994) was
cultured in an axenic medium in bioreactors, and decilia-evidence. For example, CaM has been shown to stimu-late the activity of a guanylyl cyclase whose increased tion was done by a standard procedure (Schultz et al.,
1990). The Paramecium culture and resulting cilia prepa-activity is probably correlated with various stimulatoryevents and the resulting swimming behavior (Schultz ration was kindly provided by Professor J. E. Schultz
( University of Tubingen, Germany). Frozen cilia wereet al., 1990), and application of anti-CaM drugs inhibitsexocytosis in wild-type cells ( Kerboeuf et al., 1993). mixed with a solubilization buffer (1:10 v/v), containing
2% n-octyl-b--glucopyranoside (OG), 50 mM NaCl,To further our understanding of Ca2+/CaM-regulated processes, we investigated CaM-binding pro- 5 mM EDTA, 0.02% NaN3, 50 mM Hepes (pH 7.6),
and then centrifuged at 165 000×g for 1.5 h to removeteins of Paramecium, a model system for studying vari-ous biological processes. We focused on membrane- the detergent-insoluble material, yielding a fraction
enriched with ciliary membrane proteins.bound CaM-binding proteins because there is relativelylittle knowledge on the participation of Ca2+/CaM inmembrane-associated signal transduction processes. As 2.2. 35S-labeling of CaMopposed to the abundance of information on its role inregulating more than 20 cytosolic enzymes (Crivici and 35S-labeled wild-type Paramecium CaM was produced
in Escherichia coli JM109 ( Kink et al., 1991). BacterialIkura, 1995), Ca2+/CaM has been shown to modulatethe activities of only several membrane proteins that are cultures were grown in M9 medium supplemented with
vitamins and all amino acids (Gross et al., 1984) exceptinvolved in Ca2+ homeostasis and ion channel functions.Some examples are: the plasma membrane cysteine and methionine. CaM expression was induced
in mid-log phase cultures with 1 mM IPTG for 20 minCa2+ATPase (Crivici and Ikura, 1995), the sarcoplasmicrecticulum ryanodine receptor (Tripathy et al., 1995), at 37°C in the presence of Tran35S-label (a mixture of
35S -methionine, 35S -cysteine, and various 35S-labeledthe cyclic nucleotide-gated channel in olfactory neuronsand rod outer segments (Molday, 1996), a light-acti- amino acids; ICN, Costa Mesa, CA, USA). 35S-labeled
CaM was then purified as by Kink et al. (1991). Parallelvated channel TRPL (Scott et al., 1997), the N-methyl-
23C.W.M. Chan et al. / Gene 231 (1999) 21–32
experiments substituting unlabeled -methionine and - TGRTCDATRTC; B sense, GARYARWSITTYY-TIGARGAYAA; B antisense, TTRTCYTCIARRAA-cysteine for Tran35S-label were done to estimate the
total yield of CaM and, therefore, the specific activity ISWYTRYTC; C sense, YARATHTTYAAYMMIY-ARGCICC; C antisense, GCYTRIKKRTTRAADAT-of the labeled CaM. Typical specific activity of the 35S-
labeled CaM was 1500–2000 cpm/ng protein. YTRIYC, where standard International Union ofBiochemistry codes are used. PCR was carried out ontotal Paramecium DNA with the degenerate primers in2.3. CaM blot overlayall possible combinations, using Taq DNA polymerase
CaM blot overlay experiments were performed essen- (Promega, Madison, WI, USA) with the provided buffertially as described by Evans and Nelson (1989), except supplemented with 1.5–2.0 mM MgCl2. Cycling parame-that renaturation of proteins on blots was done accord- ters were: (94°C, 2 min; 44°C, 1 min; 72°C, 1 min) foring to Hubbard and Klee (1987). The Ca2+ concen- 3 cycles; (94°C, 1 min; 44°C, 45 s; 72°C, 1 min) fortration stated throughout this paper refers to the 35 cycles and a final extension at 72°C for 5 min. PCRconcentration of free Ca2+ in the buffer. In each experi- products were screened with 32P end-labeled oligonucle-ment, 3 nM or 105 cpm/ml 35S-CaM was used. otides that were presumed to be internal to the pairsAutoradiography was performed using either Kodak used in PCR. A fragment of approx. 0.6 kb, k0.6 (aX-OMAT AR film or Phosphoimager screens PCR product amplified with A sense and C antisense,(Molecular Dynamics, Sunnyvale, CA, USA). and hybridized to both B sense and B antisense) was
thereby identified and cloned. k0.6 was labeled with 322.4. CaM affinity chromatography P with a random primer labeling kit (Rediprime,
Amersham, Arlington Heights, IL, USA) according toCaM affinity chromatography was carried out with
the manufacturer’s instructions and was then used as acommercially available CaM-Sepharose (bovine testes
probe to screen for PCM1 and its homologs in PCRCaM immobilized on Sepharose 4B, Pharmacia,
products and various DNA libraries.Piscataway, NJ, USA) since Paramecium CaM and
cDNA was reverse transcribed from purified mRNAbovine CaM are very similar and, as further demon-
(polyATtract I, Promega, Madison, WI, USA) usingstrated in the Results section, Paramecium CaM-binding
Superscript II reverse transcriptase (Stratagene, La Jolla,proteins bind bovine CaM. OG-extracted cilia were
CA, USA) with (T)24V at 46°C. The cDNA was usedloaded onto a CaM–Sepharose column pre-equilibrated
as the template in PCR with Taq DNA polymerase,with a loading buffer, consisting of 1% OG, 100 mM
using the provided buffer supplemented with 3 mMNaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.02% NaN3, MgCl2. ck0.8 was obtained and cloned after two rounds50 mM Hepes (pH 7.3). The column was washed with
of PCR. The first round of PCR was performed with aat least 10 column-volume of loading buffer before
sense strand primer GATRTAGATCATACKGGYGACaM-binding proteins were eluted by replacing 1 mM
(based on the sequence of Peptide A and taking intoCa2+ with 5 mM EGTA in the buffer. The EGTA eluate
account the nucleotide sequences of various PCM1was analyzed by sodium dodecyl sulfate–polyacrylamide
homologs in that region) and an anti-sense primergel electrophoresis (SDS–PAGE) under reducing condi-
(T )24V. The cycling parameters were : (94°C, 1 min 30 s;tions and/or CaM blot overlay experiments.
46°C, 45 s; 72°C, 2 min) for 3 cycles; (94°C, 45 s; 46°C,45 s; 72°C, 2 min) for 40 cycles and a final extension at
2.5. Peptide sequencing of PCM172°C for 5 min. The products from the first round ofPCR were then used as the template for the secondPCM1-enriched fractions from CaM-affinity chroma-round of PCR, with a sense strand primertography of OG-extracted cilia were pooled and concen-GAATAATCWTTYTTGGAAGATAA (based on thetrated, and proteins were separated by SDS–PAGE. Thesequence of Peptide B and the sequences of PCM1band corresponding to PCM1, estimated to be abouthomologs in that region) and anti-sense primer5 mg, was cut out and prepared for peptide sequencing(T )24V. The cycling parameters were: (94°C, 45 s; 46°C,according to the instructions provided by the W.M.45 s; 72°C, 2 min) for 40 cycles with a final extension atKeck Biotechnology facility at Yale University.72°C for 5 min.
The cDNA described above was also used in PCR2.6. Cloning the corresponding gene for PCM1 and itswith Pfu DNA polymerase (Stratagene, La Jolla, CA,homologsUSA), using the provided buffer (containing 2 mMMgSO4) and primers based on the DNA sequence ofTo clone the corresponding gene for PCM1, degener-
ate primers were made according to Peptides A, B and one PCM1 homolog, gk2.4. The 5∞ primer wasGAGAAGATTCTAATGGTTTGAGGG; the 3∞ primerC (obtained from peptide sequencing, see Section 3.2)
as follows (from 5∞ to 3∞): A sense, AYATHGAY- was TATTCTACTTCAATCCAGCCTCC. The cyclingparameters were: (94°C, 2 min; 62°C, 1 min; 72°C,CAYACNGGNGA; A antisense, TCNCCNGTR-
24 C.W.M. Chan et al. / Gene 231 (1999) 21–32
1 min) for 3 cycles; (94°C, 1 min; 62°C, 45 s; 72°C, shown) and were eluted from the column when Ca2+was subsequently chelated by EGTA. This EGTA-eluted1 min) for 35 cycles and a final extension at 72°C for
5 min. pck1.1 was thus obtained and cloned. fraction consistently contained a major protein with anapparent molecular mass of approx. 65 kDa, PCM1All molecular biological procedures were done using
standard protocols (Ausubel et al., 1995). All DNA (Fig. 1a). The CaM-binding activity of PCM1 was fur-ther corroborated by CaM blot overlay experimentsfragments obtained through PCR and library screening
were cloned into pBluescript KS II− (Stratagene, La (Fig. 1b). Binding of PCM1 to 35S-CaM requires aminimum of 10 mM Ca2+. Under our blot overlayJolla, CA, USA) before further analyses. DNA sequen-
cing reactions were prepared using a dye terminator conditions (10–1000 mM Ca2+ and 3 nM 35S-CaM),only high-affinity CaM-binding proteins like calcineurincycle sequencing kit (PE Applied Biosystems,
Warrington, UK ) following the manufacturer’s instruc- A and cyclic nucleotide phosphodiesterase (Crivici andIkura, 1995) bind 35S-CaM, whereas those of lowertions and were run on ABI automated sequencers
(Perkin Elmer, Norwalk, CT, USA). Analyses of all affinity such as spectrin (Crivici and Ikura, 1995), ornon-CaM-binding proteins such as the ones included inclones, including secondary structure and
hydrophilicity/hydrophobicity predictions, were aided the molecular weight standards ( low-range SDS–PAGEstandards, Biorad, Hercules, CA, USA) do not (databy the use of the DNASTAR program (Madison, WI,
USA). Potential transmembrane domains were located not shown). PCM1 is also found in purified ciliarymembrane vesicle preparations (Adoutte et al., 1980) asby manually searching for hydrophobic segments with
a peak Kyte–Doolittle hydrophobic index of at least 1.5 assayed by CaM affinity chromatography and 35S-CaMoverlay, and is absent from the cytosolic fraction asand a length of at least 17 amino acids, essentially as
described by Klein et al. (1985). Alternatively, trans- indicated by 35S-CaM overlay assays (data not shown).PCM1 is, therefore, most likely a high-affinitymembrane domain predictions were carried out with the
program TMpred (see documentation therein from Ca2+-dependent CaM-binding protein in the ciliarymembrane of Paramecium and was chosen for furtherISREC, Switzerland). Database searches were per-
formed using MPsrch (Release 2.1D, Biocomputing analyses. Starting with about 50 g of cilia and throughResearch Unit, University of Edinburgh, UK ) orWU-BLAST (BLASTP 2.0aMP, Washington University,USA) with default parameters.
The nucleotide sequences for all the clones describedin this study have been deposited in the GenBankdatabase under the accession numbers AF022488(pck1.1), AF050518 (ck0.8), AF050519 (gk2.4), andAF050520 (gk2.5).
3. Results
3.1. Identification and purification of a CaM-bindingprotein, PCM1, in the ciliary membrane of Paramecium
This project was directed towards finding new ele-Fig. 1. PCM1 is a CaM-binding protein. (a) Purification of PCM1ments in Ca2+/CaM signal transduction in an excitableusing CaM affinity chromatography. Coomassie Blue (R-250) stainedmembrane and not towards homologs of knownSDS–polyacrylamide gels loaded with various CaM affinity chroma-
Ca2+/CaM targets. Our study focused on CaM-binding tography fractions are shown. Lane 1: OG-solubilized ciliary mem-proteins in the ciliary membrane which contains impor- brane proteins, approx. 60 mg of total protein. Lane 2: void volume
(in 1 mM Ca2+), approx. 120 mg. Lane 3: wash with the loading buffertant components of signaling cascades. Furthermore,(with 1 mM Ca2+), approx. 0.5 mg. Lane 4: EGTA (5 mM ) eluate,the ciliary membrane represents about 50% of surfaceapprox. 0.3 mg. PCM1 (right-pointing arrowhead) is the major proteinmembrane area but only about 1% of total proteinseluted from the column. (b) PCM1 binds 35S-CaM. An EGTA-eluted
(Schultz et al., 1990; Adoutte et al., 1980), making it a fraction from a CaM affinity chromatography was assayed using 35S-good source for Paramecium surface membrane proteins. CaM blot overlay and the autoradiogram is shown. PCM1 (left-point-
ing arrowhead), among other proteins, binds 35S-CaM in 0.1 mMCilia were extracted with a non-ionic detergent OG toCa2+ after being immobilized on nitrocellulose membrane. The otherenrich for ciliary membrane proteins (Evans and Nelson,35S-CaM-binding activities identified here may represent other CaM-1989), and CaM-binding proteins were purified by CaM-binding proteins or proteolytic fragments of PCM1. However, since
affinity chromatography. Several proteins were retained they represent a small percentage of the total protein content of theon CaM–Sepharose in the presence of 1 mM Ca2+ (and EGTA-eluted fraction, as judged by the relative intensities of their
Coomassie-Blue staining, they were not studied further.also in the presence of 10 and 100 mM Ca2+, data not
25C.W.M. Chan et al. / Gene 231 (1999) 21–32
CaM-affinity chromatography, we obtained approx. 6 mg encodes a section of a PCM1 homolog and not PCM1itself. To gain more information on the genes encodingof PCM1.PCM1 and its homologs, k0.6 was used as a probe toscreen various DNA libraries.3.2. Peptide sequencing of PCM1
Two genomic DNA clones were found to hybridizeto k0.6. One was identified from a Paramecium genomicInitial peptide sequencing attempts suggested that
PCM1 is N-terminally blocked. Therefore, PCM1 was DNA library in EMBL4 l phage, a gift from ProfessorJ. Forney (Purdue University). The positively hybridiz-digested with trypsin and three internal peptide
sequences were then obtained as follows: Peptide A, {T ing species was narrowed down to an XbaI fragment ofapprox. 2.5 kb, gk2.5. The second genomic DNA clone,E V D I D H T G E Q A K} ; Peptide B, {F I L L E
Q S F L E D K}; Peptide C, {G V D(g) Q I F N H(t) gk2.4 (an approx. 2.4 kb NheI/PstI fragment), wasobtained from a size-fractionated Paramecium genomicQ A P}, where standard single letter amino-acid codes
are used and possible alternative amino acids are indi- DNA library. However, as explained further below,gk2.4 and gk2.5 also likely encode PCM1 homologscated in parentheses.(PCM3 and PCM4, see below) but not PCM1.
Other approaches were taken to obtain the gene for3.3. Cloning of the corresponding genes for PCM1 andits homologs PCM1. PCR was performed on cDNA with primers
based on the three sequenced peptides of PCM1 andsequence information on PCM1 homologs. A nestedUsing the peptide information available, degenerate
oligonucleotides were synthesized for PCR. As described PCR product (see Materials and methods, Section 2.6),ck0.8, was amplified using primers made to the sequencesin Materials and methods (Section 2.6), all possible
combinations of primers were attempted, and only one of Peptides A and B and the poly-dA region of thecorresponding mRNA. Another cDNA fragment,primer combination yielded a product which hybridized
to the presumed internal oligonucleotides. This PCR pck1.1, was amplified with primers based on thesequence of gk2.4 identified above. Both PCR productsproduct, k0.6, was amplified from Paramecium total
DNA with the primers A sense and C antisense and was hybridized to 32P-labeled k0.6. As explained in thefollowing sections, pck1.1 most likely codes for a portionrecognized by the oligonucleotides B sense and B anti-
sense in Southern hybridization experiments. The con- of PCM1, whereas ck0.8 encodes a section of yet anotherPCM1 homolog (PCM2, see below). The relative posi-ceptual translation of k0.6 contains Peptides A, B and
C with a few mismatches (single base pair changes; one tions of all clones mentioned above, pck1.1, ck0.8, gk2.4and gk2.5 (encoding PCM1, 2, 3 and 4, respectively),each in Peptide A and Peptide C ) that are not explained
by the degeneracy of the primers. k0.6 thus probably are shown schematically in Fig. 2.
Fig. 2. Schematic representation of pck1.1, ck0.8, gk2.4 and gk2.5. The relative positions of the two partial cDNA clones, pck1.1 and ck0.8, andthe two genomic DNA clones, gk2.4 and gk2.5, are shown, along with the probe used in Southern hybridizations (k0.6). Coding sequences (orpresumed coding sequences) are shaded, and the names of the corresponding encoded proteins are indicated in parentheses. Nucleotide positionsof the two genomic DNA clones are marked, with the first base in the assigned start codon defined as position 1, and the nucleotide sequences 5∞to that first base are labeled with negative numbers accordingly. Putative introns are denoted by , above the genomic DNA clones, and those inequivalent positions are aligned and marked by dash lines. The positions of these putative introns are 205–231, 434–460, 881–908, 1386–1410, and1671–1694 in gk2.4; 297–324, 539–564, 767–792, 1213–1237, and 1715–1740 in gk2.5. Interruptions in pck1.1 and ck0.8 mark the absence ofputative introns in these cDNA clones. Nucleotide identities to pck1.1 are: 82% for ck0.8, 77% for gk2.4, and 79% for gk2.5.
26 C.W.M. Chan et al. / Gene 231 (1999) 21–32
3.4. PCM1 and PCM2 mismatches, and is approx. 60% of the predicted lengthof PCM1 based on its apparent molecular mass.Therefore, pck1.1 is a partial cDNA clone for PCM1.The cDNA clone pck1.1 contains 1047 nucleotides
and one contiguous open reading frame (ORF), the The other cDNA clone, ck0.8, contains 778 nucleo-tides and shares 82% nucleotide identity with pck1.1conceptual translation of which corresponds to 349
residues (Figs. 2 and 3). This ORF contains all three (Fig. 2). ck0.8 has one contiguous ORF that containsPeptide B and Peptide C with two conserved amino acidsequenced peptides (Peptides A, B, and C ) with no
Fig. 3. Alignment and notable regions of PCM1–4. The amino-acid alignment of PCM1–4 is shown. Identical residues are marked in black, andconserved substitutions are lightly shaded. Amino-acid identities among the four proteins, with that of PCM1 as the standard, are: PCM2, 85%;PCM3, 89%; PCM4, 87%. The period at the end of PCM2 and PCM3, respectively, indicates the stop codon in each of the corresponding DNAclones. Notable regions are indicated as follow. The putative b/c-crystallin domain is marked by a shaded bar below, and the two types of cysteine-rich regions are marked with double-headed arrows (The Type I cysteine-rich region is C-terminal to Type II ). Peptides A, B, and C in PCM1(the three sequenced peptides, from N-terminal to C-terminal ), and their counterparts in PCM2–4, are indicated by dash lines underneath. Allfour PCM proteins contain ‘D’ in residue three and ‘H’ in residue eight of Peptide C and, therefore, the possible alternative amino acids in PeptideC according to peptide sequencing analysis have not been found. The positions of putative BAA helices are: residues 17–40 and 208–233 for PCM1,residues 102–127 for PCM2, residues 209–232 and 399–424 for PCM3, and residues 57–80, 311–334 and 501–526 for PCM4. Standard single-letter amino acid codes are used. Dash lines indicate gaps introduced to optimize the alignment. Residue positions are indicated on the side. Thealignment is generated with the Clustal method of MegAlign (DNASTAR) with default parameters.
27C.W.M. Chan et al. / Gene 231 (1999) 21–32
substitutions (Fig. 3). This ORF also contains the stop entire coding sequence for PCM3 is contained withingk2.4.codon TGA, and encodes a polypeptide of 243 amino
acids. Its deduced protein is 85% identical to PCM1 The longest possible ORF in gk2.5 has 1861 nucleo-tides (Fig. 2). This ORF begins with a start codon (theand, therefore, the entire predicted protein may also be
homologous to PCM1. This suggests that ck0.8 encodes nearest in-frame stop codon is 63 nucleotides upstream),has an A/T composition of 62%, and is preceded by athe C-terminal approx. 40% of a PCM1 homolog. We
call this protein PCM2. region of higher % A/T (77% for 657 nucleotides in the5∞). Therefore, this ORF encompasses the N-terminal577 amino acids of its corresponding protein (Fig. 3).3.5. PCM3 and PCM4If we assume that all members of the PCM family havehomologous C-termini, as suggested above, then gk2.5Nucleotide sequences of gk2.4 and gk2.5 are very
similar to each other and to that of pck1.1 and ck0.8, covers approx. 90% of the complete coding sequence(or that gk2.5 is approx. 220 nucleotides short of the 3∞sharing approx. 80% identities in their corresponding
regions (Fig. 2). To determine the longest possible ORFs end). This also implies that the entire protein consistsof 642 amino acids, corresponding to approx. 72 kDa.in these two sequences, we considered their distributions
of A/T and the presence of putative introns. The However, if we omit the first putative intron, the pre-dicted protein of the resulting shorter ORF containsParamecium genome contains a high percentage of A/T
nucleotides. However, coding sequences are typically 552 amino acids, or approx. 62 kDa, making it evenmore similar in size to PCM1 and PCM3. This possibleless A/T rich (approx. 65%) than their surrounding,
non-coding regions (approx. 80%) (Elwess and Van variation in the N-terminal sequence of the deducedprotein of gk2.5 does not affect its high degree ofHouten, 1997). Introns can be predicted by the presence
of intron consensus, characterized by the length and similarity to other members of the PCM family: It is87% identical to PCM1, and contains Peptides A, B,A/T content of intron areas and specific sequences that
border the introns (Russell et al., 1994). We also took and C with a few conserved substitutions (Fig. 3).Therefore, gk2.5 seems to code for a fourth member ofinto account that homologous genes may have conserved
exon–intron boundaries, a phenomenon previously the PCM family, PCM4.observed in Paramecium (Russell et al., 1994). The lackof putative introns but conservation of the surrounding 3.6. The PCM familysequences in the corresponding regions of pck1.1 andck0.8 further supports our assignment of introns The existence of at least four to five members in the
PCM family is supported by results from genomic(Fig. 2). Conceptual translations thus obtained fromthe two genomic fragments yield predicted proteins that Southern hybridization experiments. When Paramecium
genomic DNA was digested to completion with variousare similar to PCM1 and PCM2, and are close to theapparent molecular mass of PCM1, as explained in more restriction enzymes and then probed with 32P-labeled
k0.6 (a section of a putative PCM1 homolog), four ordetail below.The longest ORF in gk2.4 consists of 1754 nucleotides more bands hybridized (Fig. 4a). However, Northern
hybridization analysis on mRNA with k0.6 as the probe(Fig. 2), starting with the initiation codon (the nearestin-frame stop codon is three nucleotides upstream) and revealed only one major hybridizing species at approx.
1.9 kb (Fig. 4b). Assuming the length of theending with the stop codon. The stop codon is in aposition equivalent to that in ck0.8, and very close to untranslated region is minimal, this suggests that the
majority of the expressed proteins from the PCM familythe last predicted amino acid of pck1.1 (Fig. 3), suggest-ing that all members of the PCM protein family have are approx. 70 kDa. These approx. 70 kDa PCM pro-
teins can consist mainly of PCM1, as suggested by oursimilar C-termini and that pck1.1 codes for theC-terminal approx. 60% of PCM1. This longest ORF biochemical data, or they can contain various PCM
proteins very similar in sizes, a notion consistent withhas an A/T content of 63% and is surrounded by regionsof higher % A/T (approx. 80% A/T for about 70 our assignment of ORFs and the predicted proteins
thereof.nucleotides in the 5∞ and 76% for 428 nucleotides in the3∞). Therefore, gk2.4 most likely encompasses the entirecoding region of a protein of 540 amino acids, corre- 3.7. Predicted amino-acid sequences and corresponding
protein motifs of the PCM familysponding to approx. 61 kDa, which is very similar tothe apparent molecular mass of PCM1 of approx.65 kDa. The predicted amino-acid sequence of gk2.4 is Database searches with sequences of the PCM family
did not reveal any strong candidates for homologs as of89% identical to PCM1, and contains Peptides A, B,and C with a few conserved substitutions (Fig. 3). The writing, suggesting that they are novel proteins.
However, they share similar protein motifs, with allpresent data support the notion that gk2.4 encodes athird member of the PCM family, PCM3, and that the members of the family expected to contain putative
28 C.W.M. Chan et al. / Gene 231 (1999) 21–32
Ikura, 1995). PCM1 is a CaM-binding protein andcontains at least two putative BAA helices, residues 17–40 and 208–233. These two putative BAA helices areconserved correspondingly in PCM3 (residues 209–232and residues 399–424) and PCM4 (residues 311–334and residues 501–526), and PCM2 appropriately con-tains the more C-terminal one (residues 102–127)(Fig. 3). In addition, PCM4 appears to contain anotherpotential BAA helix (residues 57–80).
Two types of cysteine-rich regions are found in thePCM proteins. The Type I cysteine-rich region contains20% cysteines in a window of 30 amino acids, and is inequivalent positions in PCM1 (residues 68–97), PCM3(residues 259–283) and PCM4 (residues 361–390),respectively (double-headed arrows in Fig. 3). PCM3and PCM4 contain an additional type of cysteine-richregion that is N-terminal to the first (residues 83–91 ofPCM3 and residues 185–201 of PCM4). This Type II
Fig. 4. Southern and Northern hybridizations showing the number of cysteine-rich region contains four cysteines in a windowmembers in the PCM family and the size of their mRNA. (a) Southern of 17 amino acids (or approx. 24% cysteines). Thehybridization. About 10 mg each of Paramecium genomic DNA was
position and spacing of the cysteines in the two typesdigested with the restriction enzymes ClaI (Lane 1) and BglII (Laneof cysteine-rich regions are absolutely conserved. The2), separated by agarose gel electrophoresis, transferred onto nitrocel-
lulose membrane, and hybridized to 32P-labeled k0.6. The resulting presence of conserved cysteine-dense regions in proteinsautoradiograph shows four or more hybridizing bands ( judging from that are not particularly cysteine-rich (4% or less cysteinethe relative intensities of bands, the second band from the top in Lane residues overall ) argues for their functional significance.2 can be interpreted as a doublet), suggesting that there are at least
Another kind of protein motif present in the PCMfour members in the PCM family. This interpretation is consistent withfamily is the b/c-crystallin domain. b- and c-crystallinsour cloning data which include sequences of four members of the PCM
family (Their sequences indicate that ClaI and BglII do not have belong to the same protein superfamily and were discov-restriction sites within the regions that hybridize to k0.6). (b) Northern ered as two of the major classes of lens proteins inhybridization. Approx. 15 mg of oligo-dT purified mRNA was sepa- vertebrates. However, some b-crystallins are expressedrated by agarose gel electrophoresis in the presence of formamide,
at lower levels in tissues other than the lens, such as thetransferred onto uncharged nylon membrane, and hybridized to 32P-retina. Non-lens members of the b/c-crystallin superfam-labeled k0.6. The autoradiograph shows one major hybridizing band
at approx. 1.9 kb, which may represent the mRNA of the major ily have also been found in a variety of organisms (Rayexpressed protein from the PCM family or several mRNA species of et al., 1997). All members of the family share a conservedvery similar sizes. domain containing two homologous motifs. Each of
these motifs consists of four b strands folded into acharacteristic ‘Greek key’ pattern (Bagby et al., 1994).transmembrane domains, potential basic amphiphilic
alpha (BAA) helices, cysteine-rich regions, and putative The C-terminal (or presumed C-terminal ) regions of allmembers of the PCM family (shaded bars in Fig. 3)b/c-crystallin domains (Fig. 3 and see below).
Members of the PCM family have very similar pre- share significant sequence similarities with members ofthe b/c-crystallin family. For example, the amino-aciddicted hydrophilicity/hydrophobicity profiles. Two of
the hydrophobic segments in PCM1 can be considered identities between the C-terminal regions of the PCMfamily and various mammalian c-crystallins are approx.as potential transmembrane domains (residues 89–108
and 139–155), and they are also conserved in other 30%, similar to the sequence identities shared betweenb- and c-crystallins. Furthermore, the alignment of thesemembers of the family (residues 33–49 in PCM2, resi-
dues 280–299 and 330–346 in PCM3, and residues 382– C-termini with selected members of the b/c-crystallinfamily (Fig. 5) shows that they contain all the conserved401 and 432–448 in PCM4). The unique N-terminal
region of PCM4 contains an extra hydrophobic segment residues important for the structural integrity of theb/c-crystallin domain (Y6, Y10, G13, and S36 in each(residues 4–30) that may represent an additional trans-
membrane segment. motif of PCM1, shaded residues in Fig. 5). Of particularimportance is the conservation of G13 because it playsThe PCM family also contains several potential BAA
helices. BAA helical regions tend to form a-helices with a crucial role in maintaining the characteristic b hairpinformed by the first two b strands of the ‘Greek key’positively charged amino acids facing one side of the
helix and hydrophobic ones on the other side, and they motif (Bagby et al., 1994). These putative b/c-crystallindomains of the PCM family are also predicted to behave been found to be the major CaM-binding determi-
nant in a variety of CaM-binding proteins (Crivici and mainly b in secondary structure (e.g., Protean of
29C.W.M. Chan et al. / Gene 231 (1999) 21–32
Fig. 5. b/c-Crystallin domains. The alignment of the putative b/c-crystallin domains of the PCM family with the corresponding domains of selectedmembers of the b/c-crystallin family is shown. Based on published structural data and models, the first four entries are aligned manually accordingto structurally important residues (shaded), and residues that make up the four known b sheets (underlined). Conserved core hydrophobic residues,marked with filled circles above ($), are also taken into consideration. The C-terminal regions of PCM1–4 are then aligned accordingly. Thealignment shows that every member of the PCM family contains all of the conserved elements of typical b/c-crystallin domains. Moreover, residues260–266, 293–297, 303–308, 317–319, and 332–336 of PCM1 (and corresponding residues from PCM2–4) are predicted to form b sheets by varioussecondary structure prediction programs (e.g., PROTEAN from DNASTAR, PSSP from the Baylor College of Medicine, and PHD from EMBL-Heidelberg), and they correspond to assigned b sheets of other members of the b/c-crystallin family. c-Crystallin II and b-crystallin B2 are majorlens proteins in bovine, Protein S is a major spore coat protein in Myxococcus xanthus (a soil bacterium), and Spherulin 3A is a major encystmentprotein in Physarum polycephalum (a slime mold). Dash lines indicate gaps introduced to optimize alignment. Residue positions are indicated onthe side.
DNASTAR, PSSP from the Baylor College of Medicine PCM1 is consistently associated with the membranefraction despite being prepared by different methods.and PHD from EMBL-Heidelberg) and can indeed be
modeled as several anti-parallel b sheets (data not Moreover, PCM1 remains associated with the membranefraction after high salt washes (up to approx. 0.4 Mshown; SWISS-MODEL at ExPASy), consistent with
structural data on other members of the b/c-crystallin NaCl ), a procedure typically used to separate peripher-ally-associated membrane proteins from membrane-family (Bagby et al., 1994; Rosinke et al., 1997).bound proteins (data not shown), indicating that PCM1is an integral membrane protein, or is tightly associatedwith the membrane.4. Discussion
PCM1 is one of the major CaM-binding proteins inthe ciliary membrane and binds CaM at near physiologi-4.1. PCM1: a CaM-binding membrane protein in cilia of
Paramecium cal Ca2+ concentrations (approx. 10 mM). Its affinityfor CaM is comparable to other high affinity CaM-binding proteins such as calcineurin and cyclic nucleo-Our knowledge of Ca2+/CaM-regulated processes
through membrane-associated proteins lags behind that tide phosphodiesterase (see Section 3.1). Although thelocation of the CaM-binding site(s) within PCM1 hasvia their cytosolic counterparts. Therefore, it is impor-
tant to identify and characterize membrane-bound parti- yet to be experimentally determined, present datastrongly suggest that PCM1 is a physiologically relevantcipants of Ca2+/CaM-modulated pathways. In the
model system Paramecium, we have identified and puri- CaM target protein in vivo.fied a Ca2+-dependent CaM-binding protein, PCM1,from a ciliary membrane-enriched fraction. (Although 4.2. The PCM protein familywe purified PCM1 from the ciliary membrane, PCM1may not be localized exclusively to this membrane and Based on the peptide sequences of PCM1, we cloned
the genes for a family of PCM proteins, PCM1–4. Themay indeed be present in other surface membranes.C.W.M. Chan, unpublished results). PCM1 was also protein encoded by k0.6 may also belong to the PCM
family, although at present we do not know whether itpresent in highly purified ciliary membrane vesicles,obtained by physically disrupting the surface membrane shares the same protein motifs found in all other mem-
bers of the family. The results from genomic Southernfrom cilia and subsequently isolating the membranevesicles by centrifugation in a sucrose gradient (Adoutte hybridization experiments support the existence of at
least four members in the PCM family (Fig. 4a). Theet al., 1980), but not in the cytosolic fraction. Therefore,
30 C.W.M. Chan et al. / Gene 231 (1999) 21–32
three sequenced peptides of PCM1 is found in the the two homologous b/c-crystallin domains interactthrough some of their component b strands, contributingconceptual translation of only one of the four DNA
clones described in this study (pck1.1), whereas the to the characteristic structural stability of c-crystallinsand dimer formation among various b-crystallinsother three clones contain various conserved substitu-
tions in their corresponding regions. Therefore, pck1.1 (Bagby et al., 1994). Dimers of b-crystallins can furtherassociate to form different populations of homo- andis the partial cDNA clone for PCM1, covering approx.
60% of the predicted length of the protein. Moreover, hetero-oligomers under various conditions (Slingsby andBateman, 1990). Non-lens members of the b/c-crystallinwe have the partial coding sequences for PCM2 and
PCM4 (covering approx. 40% and approx. 90% of their family, such as Protein S in Myxococcus xanthus andSpherulin 3A in Physarum polycephalum, are also knownrespective proteins), and the entire gene for PCM3.
The coding region for PCM3 is approx. 1.8 kb, in to form homomultimers (Rosinke et al., 1997; Bagbyet al., 1994). We propose that in PCM1–4, theirgood agreement with the approx. 1.9 kb mRNA species
detected in our Northern hybridization analysis C-terminal regions are folded into ‘Greek key’ motifsthrough which they interact with each other and multi-(Fig. 4b). Since all PCM proteins are predicted to be
similar in sizes, the approx. 1.9 mRNA band may merize, similar to other members of the b/c-crystallinsuperfamily. Various members of the PCM family (andrepresent the expression of several PCM proteins, includ-
ing the two for which cDNA clones have been obtained, possibly other proteins bearing similar b/c-crystallinmotifs) may be co-expressed and can then form a varietyPCM1 and PCM2. On the other hand, since PCM1 is
likely to have the highest expression level among various of homomultimers and even heteromultimers. If theexpression of PCM family members is alternativelymembers of the PCM family, as evidenced by our
biochemical and peptide sequencing data, this mRNA regulated, different populations of homomultimers maybe formed according to their modes of regulation.band may consist mainly of the message for PCM1.
All members of the PCM family have very similar The PCM proteins of Paramecium are part of anexpanding group of non-lens members of the b/c-crystal-conceptual translations (approx. 85% identical ). They
all contain conserved protein motifs, potential CaM- lin superfamily, and they seem to have a general role ofmanaging cellular differentiation and morphologicalbinding domains and putative transmembrane segments.
No candidates for homologs of the PCM family in other changes. For instance, Protein S and its homolog ProteinS1 from M. xanthus (Bagby et al., 1994) and Spherulinorganisms have yet been reported. In particular, the
PCM family of proteins do not share significant sequence 3A from P. polycephalum (Rosinke et al., 1997) areinduced under adverse environmental conditions thatsimilarities with the handful of membrane-bound CaM-
binding proteins identified to date, nor with other CaM- lead to dormancy. EDSP (epidermis differentiation-specific protein) from Cynops pyrrhogaster and AIM1dependent enzymes (Crivici and Ikura, 1995; Tripathy
et al., 1995; Molday, 1996; Scott et al., 1997; Xia et al., (absent in melanoma) from a model of human melanoma(Ray et al., 1997) are expressed in tissues with ectoder-1998; Zhang et al., 1998). Furthermore, they do not
have any identifiable catalytic domain or substrate- mal origins, are possibly associated with the cytoskeletonand may have roles in managing cell morphology andbinding site of any characterized enzyme. They also do
not contain any recognizable ion channel structure, nor shape. Even in lens, b- and c-crystallins are specificallyexpressed in differentiating and elongating fiber cellsdo they belong to any of the known classes of membrane-
associated structural proteins. Thus, the genes identified which are undergoing large changes in cellular architec-ture and composition, and it has been suggested thathere seem to be truly novel, and likely encode a new
family of CaM-binding membrane proteins. these proteins may have functions other than theirstructural role (Ray et al., 1997). By inference, the PCMproteins of Paramecium may belong to a class of4.3. Protein motifs for protein–protein interaction and
multimer formation, and implications for Ca2+/CaM Ca2+-sensing molecules that respond to changing envi-ronmental conditions and/or developmental stages. Onesignal transductionhypothesis is that during increases in internal Ca2+concentration, CaM binds to PCM1 (and may also bindThe PCM family contains recognizable protein motifs
that may give clues to their function(s). All PCM to PCM1 homologs), which then leads to a change inthe conformation of the PCM-containing protein com-proteins are predicted to contain putative b/c-crystallin
domains at their C-terminal (or presumed C-terminal ) plex or its state/composition of multimerization. Thisthen serves as a mechanism for regulating the function(s)regions (Figs. 3 and 5). These C-terminal regions share
significant sequence similarity with other members of of PCM-containing multimers and subsequently bring-ing about appropriate physiological changes. Inthe b/c-crystallin superfamily, contain all the conserved
and structurally important residues for the domain, and Paramecium, Ca2+/CaM is known to (or likely) partici-pate in a wide range of processes, including cellularare predicted to form several b sheets (Section 3.7).
Structural studies on b- and c-crystallins indicate that morphogenesis, trichocysts discharge, and swimming
31C.W.M. Chan et al. / Gene 231 (1999) 21–32
Fig. 6. Cysteine-rich regions. The alignment of the consensus sequence of the Type I cysteine-rich regions in the PCM family with the consensussequence of EGF-like domains is shown. Both consensus sequences are given in the PROSITE format, where ‘x’ stands for any amino acids, ‘a’stands for aromatic residues, the figure in (n) indicates the number of residues, and the figures in (n,n) indicate the range of the number of residues.Shaded boxes mark the regions where the two consensus sequences agree with each other, showing that the Type I cysteine-rich regions in thePCM family resemble EGF-like domains. Of the two glycines given in the consensus sequence of EGF-like domains, at least one is usually present,and each of the Type I cysteine-rich regions contains one glycine correspondingly.
behavior, and the PCM family of proteins may be an proteins with characteristic protein–protein interactiondomains, and they participate in Ca2+/CaM signalintegral part of these signaling/response systems.
The Ca2+/CaM-dependent signaling and response transduction.The PCM proteins represent new membrane-associ-pathway through the PCM proteins may be further
modulated by other domains in these proteins. One such ated transducers of Ca2+/CaM-dependent cascades, andit is important to further analyze these proteins, particu-possibility is the cysteine-rich regions. The Type I cyste-
ine-rich region contains six conserved cysteines in a larly with regard to how they propagate theCa2+/CaM signal. Biochemical analyses concerning thewindow of 30 amino acids (20% cysteines). The Type II
cysteine-rich region contains four conserved cysteines in details of CaM binding to PCM1 and its homologs andthe conditions for multimerization are most interesting.a total of 17 residues (approx. 24% cysteines). The
conservation of cysteine-dense regions in a protein Immunological and Western blot studies using antibod-ies generated against the PCM family of proteins willfamily that is otherwise not cysteine-rich suggests that
these regions are of functional significance. Of particular provide information on their expression and sub-cellularlocalization, and whether these parameters change withinterest is the Type I cysteine-rich region, which is
reminiscent of an epidermal growth factor (EGF)-like varying environmental and cellular conditions. InParamecium, we can also investigate the in vivo func-domain, as shown in Fig. 6. The EGF-like domain was
first found in EGF and subsequently in the extracellular tion(s) of the PCM family by altering their expressionlevels, which can be achieved through microinjectingdomains (or putative extracellular domains) of a variety
of membrane-bound or secreted proteins. In EGF the appropriate DNA fragments (Haynes et al., 1996; Meyerand Duharcourt, 1996; Ruiz et al., 1998).six cysteines are disulfide-bonded in a (1–3, 2–4, 5–6)
pattern, resulting in a tri-stranded b sheet structure Ca2+/CaM is vital in regulating membrane-associatedsignal transduction processes. However, the details on(Rebay et al., 1991). The function of the domain is still
under investigation. In certain cases, however, it has how Ca2+/CaM and its associated proteins participatein these signaling cascades await more extensive studies.been shown to mediate protein–protein interaction, as
in the example of EGF binding to its receptor, and cell On-going and future research of the PCM family willenhance our understanding of Ca2+/CaM signal trans-surface interactions of the neurogenic proteins Notch
and Delta in Drosophila (Rebay et al., 1991). It is duction in Paramecium, and will likely shed light on thesubject in other organisms as well.possible that the six cysteines in the Type I cysteine-rich
region are disulfide-bonded in a pattern analogous tothat in EGF, implying a similar protein folding patternand even a role in interacting with yet unidentified
Acknowledgementsligands or proteins on the cell surface. The resultingtertiary structure and/or interaction with ligands and
We thank Professor J.E. Schultz for providingproteins may be important for the activities of PCM-Paramecium culture and the resulting cilia preparation,containing protein complexes.and Professor J. Forney for providing the Parameciumgenomic DNA library. We also thank L. Olds for aidin preparing the figures, and the staff of the W.M. Keck5. Summary and future directionsBiotechnology Center for their expert technical service.This work is supported by NIH GM22714, GM36386,We have discovered a new family of membrane-and the W.F. Vilas Trust.bound, Ca2+-dependent CaM-binding proteins, and
have obtained the genes for four members of this family,PCM1–4. Although only the gene for PCM3 is complete,the partial coding sequences for other members of the ReferencesPCM family also contain all the protein motifs sharedamong the family. Thus, current data have revealed the Adoutte, A., Ramanathan, R., Lewis, R.M., Dute, R.R., Ling, K.Y.,
Kung, C., Nelson, D.L., 1980. Biochemical studies of the excitableessence of the PCM family: a collection of closely related
32 C.W.M. Chan et al. / Gene 231 (1999) 21–32
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