C. elegans spermatogenesis employs lysosome-related fibrous body–membranous organelles (FB–MOs) for transport of many cellular components. Previous work showed that spe-10 mutants contain FB–MOs that prematurely disassemble, resulting in defective transport of FB components into developing spermatids. Consequently, spe-10 spermatids are smaller than wild type and contain defective FB–MO derivatives. In this article, we show that spe-10 encodes a four-pass integral membrane protein that has a DHHC–CRD zinc-finger motif. The DHHC–CRD motif is found in a large, diverse family of proteins that have been implicated in palmitoyl transfer during protein lipidation. Seven spe-10 mutants were analyzed, including missense, nonsense, and deletion mutants. An antiserum to SPE-10 showed significant colocalization with a known marker for the FB–MOs during wild-type spermatogenesis. In contrast, the spe-10(ok1149) deletion mutant lacked detectable SPE-10 staining; this mutant lacks a spe-10 promoter and most coding sequence. The spe-10(eb64) missense mutation, which changes a conserved residue within the DHHC–CRD domain in all homologues, behaves as a null mutant. These results suggest that wild-type SPE-10 is required for the MO to properly deliver the FB to the C. elegans spermatid and the DHHC–CRD domain is essential for this function.
CAENORHABDITIS elegans spermatogenesis utilizes the vesicular trafficking pathway to help establish cellular polarity during differentiation. Each primary spermatocyte undergoes meiosis to produce four haploid spermatids (Figure 1A). Spermatids selectively retain required components, while unnecessary components accumulate in the anucleate residual body. The vesicular trafficking pathway produces fibrous body–membranous organelles (FB–MO) that play a major role during this asymmetric cytoplasmic partitioning (reviewed by L'Hernault 1997; L'Hernault 2005).
The fibrous bodies (FBs) and membranous organelles (MOs) form in close association as the primary spermatocyte develops (Figure 1C). Each MO that arises from the Golgi has a double-layered membrane “cup” surrounding major sperm protein (MSP) fibers that compose its associated FB (Figure 1, C1 and C2), and many FB–MO complexes are found in each primary spermatocyte (Figure 1A). FB–MOs segregate to each budding spermatid simultaneous with completion of meiosis (Roberts et al. 1986), while ribosomes, actin, most of the tubulin, and various membranes are discarded in the residual body (Ward 1986). MO membranes withdraw from the FBs as spermatids separate from the residual body (Figure 1C3), and FBs depolymerize into constituent MSP dimers (Klass and Hirsh 1981) that disperse throughout the cytoplasm (Roberts et al. 1986). During conversion of spermatids into spermatozoa, MSP dimers concentrate in the pseudopod (Figure 1A; Ward and Klass 1982) and polymerize into the cytoskeleton that mediates cell motility (Italiano et al. 2001). MOs localize under the spermatozoan cell surface where they fuse with the plasma membrane and form a permanent fusion pore. This exocytotic event releases MO contents onto the cell surface, which is necessary for spermatids to become functioning spermatozoa (Figure 1C5).
Analyses of over 60 spermatogenesis-defective (spe) mutants in C. elegans have identified several mutants that disrupt the morphogenesis of the FB–MOs (reviewed by L'Hernault 1997; L'Hernault 2005). Six mutants that have been studied in detail are spe-4, spe-5, spe-6, spe-10, spe-17, and spe-39. spe-39 mutant spermatocytes, which display the earliest defects, do not contain the MO portion of the FB–MO. Instead, spe-39 mutants accumulate small vesicles, have their FBs scattered around the cytoplasm, and usually arrest development as terminal spermatocytes containing four haploid nuclei rather than becoming four spermatids (Zhu and L'Hernault 2003). spe-6 mutants fail to assemble MSP dimers into FB filaments but instead distribute them throughout the spermatocyte cytoplasm. The developing MOs become vacuolated and are randomly located throughout the cytoplasm (Varkey et al. 1993). Both spe-4 and spe-5 mutants have cytokinesis defects so morphogenesis results in a terminal spermatocyte, as described above for spe-39 (L'Hernault et al. 1988; Machaca and L'Hernault 1997). The spe-4 mutant terminal spermatocyte contains abnormal FB–MO complexes with vacuolated MOs that are not associated with the FBs (L'Hernault and Arduengo 1992). The spe-5 mutant spermatocytes also have vacuolated MOs, though a few spe-5 spermatocytes complete the budding process, generating mutant spermatids with vacuolated MOs (Machaca and L'Hernault 1997). In spe-17 mutants, the FB–MOs assemble but membranes are studded with ribosomes (Shakes and Ward 1989), a phenomenon that is never seen in wild-type spermatocytes (Wolf et al. 1978; Ward et al. 1981). Although these FB–MOs segregate to the budding spermatids, many of the MOs fail to fuse with the spermatid cell surface. In spe-10 mutants, FB–MOs disassemble prior to the completion of spermatid budding. Resulting MOs segregate to the budding spermatids where they become vacuolated and do not fuse with the cell surface (Shakes and Ward 1989). In contrast, the FBs are placed in the residual body and do not segregate to the budding spermatid as they do in wild type (Figure 1B). These FBs can associate with the residual body plasma membrane and bud off as small FB cytoplasts. The resulting spe-10 mutant spermatids are smaller than wild type (N2), have abnormally short pseudopods, and have never been seen to move by the crawling that characterizes wild-type spermatozoa (Shakes and Ward 1989).
In this article, we show that spe-10 encodes a sperm-specific transmembrane protein that contains a zinc-finger motif called NEW1 (Bohm et al. 1997), DHHC–CRD (cysteine-rich domain) motif (Putilina et al. 1999), or most recently, zf-DHHC (Pfam accession no. PF01529). The DHHC–CRD proteins are a large, diverse protein family (at least 346 members) found throughout eukaryotes and can have 0–11 transmembrane (TM) domains. SPE-10 is found in a subfamily that all have four TM domains. We have raised an antiserum that shows SPE-10 resides mostly in FB–MOs and that a spe-10 knockout mutant has no detectable signal. Biochemical data from others suggest that the DHHC–CRD domain catalyzes transfer of palmitate lipid to substrate proteins. Throughout eukaryotes, palmityolation plays an important regulatory role during many signaling events and the trafficking of vesicular proteins (reviewed by Smotrys and Linder 2004). This suggests that SPE-10 could be regulating one or more critical palmitoylation events required for proper interaction of the FB with the MO during spermatogenesis.
MATERIALS AND METHODS
Strains, culture, and nomenclature:
C. elegans var. Bristol (N2) was the wild-type strain used in all experiments. Culturing, manipulation, and genetic analysis of worms were performed as described (Brenner 1974), and standard C. elegans nomenclature was employed (Horvitz et al. 1979). The following genetic markers, mutations, deficiencies, and balancer chromosomes were used: fem-3(q23gf)IV (Barton et al. 1987), fem-1(hc17)IV (Nelson et. al. 1978), spe-10(hc104)V (Shakes and Ward 1989), sma-1(e30)V (Brenner 1974), him-5(e1490)V, him-8(e1489)IV (Hodgkin et al. 1979), sDf35 V (McKim et al. 1988), mIs10 V (K. Liu and A. Fire, unpublished), nT1[unc-?(n754) let-?]IV; +/nT1 V (also known as DnT1; Ferguson and Horvitz 1985) and DnT1[qIs50] (F. H. Markussen and J. Kimble, unpublished results).
Origin of spe-10 alleles:
Males were mutagenized with either 50 mm ethyl methanesulfonate (EMS) (Brenner 1974) or 1.75 mm N-ethyl-N-nitroso urea (ENU) (Anderson 1995) for 4 hr and mated in an F1 noncomplementation screen with either homozygous spe-10(hc104) or spe-10(eb64) hermaphrodites that had been raised at 25°. For the EMS screens, mIs10 V males were mutagenized, and 4857 of the L4 F1 hermaphrodites they sired were picked to separate plates. For the ENU screens, N2 or mIs10 V males were mutagenized, and 10,150 L4 F1 hermaphrodites they sired were picked to separate plates. Both screens were performed at 25° and self-sterile, oocyte-laying individuals were identified the following day. These screens resulted in the EMS-induced eb112 and eb118 spe-10 alleles and ENU-induced eb64, eb105 and eb106 spe-10 alleles. The spe-10(ok1149) allele was an ultraviolet/trimethyl psoralen (UV/TMP) induced knockout provided by the C. elegans Knockout Consortium (see http://celeganskoconsortium.omrf.org/). The spe-10 alleles induced on mIs10 chromosomes, which are associated with green-fluorescent protein (GFP) expression (Chalfie et al. 1994), were crossed to N2 and nonGFP Spe recombinants were recovered. All the spe-10 alleles were outcrossed to N2 and then balanced over DnT1 or DnT1[qIs50], which confers a dominant GFP+ signal, to facilitate subsequent genetic and phenotypic analysis.
Individual hermaphrodites were picked to separate plates and transferred daily, which allowed counting the number of progeny and oocytes until the worms stopped laying (L'Hernault et al. 1988). Temperature-sensitive periods were determined as previously described (Hirsh and Vanderslice 1976). Protocols for sperm isolation from males and their in vitro activation with Pronase were as described previously (L'Hernault and Roberts 1995), except that modified SM containing 10 mm glucose was the sperm medium employed (Machaca et al. 1996). An Olympus BX60 compound microscope equipped with either a Plan-Apo 1.4 NA 60× or a 1.35 NA 100× oil-immersion objective lens was used to view sperm. Twelve-bit images were captured with a SensiCam digital camera (Cooke, Auburn Hills, MI) using Image-Pro PLUS (Media Cybernetics, Silver Spring, MD) and VolumeScan 3.1 (VayTek, Fairfield, IA) software. Twelve-bit images were converted to eight bits in Image-Pro PLUS and compiled using Canvas 9 (ACD Systems, Miami) or PhotoShop 7.0 (Adobe Systems, San Jose, CA).
Transgenic rescue of spe-10:
spe-10 was previously mapped between unc-42 V and sma-1 V, right of mDf1 and left of ctDf1 under sDf35 (Shakes and Ward 1989). Overlapping yeast artificial chromosome (YAC) and cosmid clones corresponding to this region (Coulson et al. 1995) were micro-injected into adult +/DnT1 IV; spe-10(hc104) sma-1(e30)/DnT1 V or N2 hermaphrodites as previously described (Mello and Fire 1995). Recombinant cosmid or YAC DNA was co-injected with either pRF4, which contains the rol-6(su1006) dominant mutation (Mello and Fire 1995), or pPD118.20 (1997 Fire Lab Vector Kit), which is a plasmid that contains the myo-3 body-wall muscle promoter driving GFP. YAC and cosmid clone DNAs were prepared for micro-injection as previously described (Browning and Strome 1996). Restriction fragments derived from cosmid AC3 were micro-injected to localize the spe-10 ORF within this cosmid.
A 3,332 bp SalI/SacI genomic restriction fragment derived from cosmid AC3 was cloned into Bluescript KS+ (Stratagene, La Jolla, CA) to create pSL41. A NotI site was created between the penultimate codon and the stop codon of pSL41 by site-directed mutagenesis with the Gene Editor kit (Promega, Madison, WI) to create pSL42. The pGETP1 plasmid (Tyers et al. 1992) was digested with NotI, and a 111-bp restriction fragment that encodes its three sequential hemaglutinin (HA) epitopes was gel purifed. This restriction fragment was ligated into NotI digested pSL42 to create pSL43. pSL43 encodes the SPE-10 amino acid sequence with GGRIFYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAA QCGR added to the C-terminal end; the individual HA epitopes are underlined. A simple repeat extrachromosomal array, ebEx423, was made from this construct, crossed into spe-10(ok1149), and assayed for self-fertility (Table 1). The SalI/SacI insert fragment from pSL43 was subcloned into a unique, blunted ApaI site of the pPD118.20 vector (1997 Fire Lab Vector Kit) to create pSL44. pPD118.20 has myo-3 promoter sequence 5′ and let-858 untranslated sequence 3′ to GFP coding sequence. pSL44 was restriction digested with PsiI and used to create “complex” array transgenes (Kelly et al. 1997) by co-injecting it together with PvuII restricted genomic DNA that was prepared from spe-10(ok1149) homozygotes grown at 16°. One of these transgenes, ebEx496, was crossed into spe-10(ok1149) hermaphrodites and assayed for self-fertility (Table 1).
Total RNA was isolated from him-5(e1490), fem-3(q23 gf), and fem-1(hc17) worms as described previously (Reinke et al. 2000). The him-5(e1490) strain produces approximately 33% male self-progeny (Hodgkin et al. 1979), and these males were purified from cultures as described previously (L'Hernault and Roberts 1995). fem-3(q23 gf) worms are hermaphrodites with a germ line that only produces sperm when they are grown at 25° (Barton et al. 1987). fem-1(hc17) worms are hermaphrodites with a germ line that only produces oocytes when the worms are grown at 25° (Nelson et al. 1978). Approximately 20 μg of RNA was loaded in each lane of a 1.4% formaldehyde agarose gel, subjected to electrophoresis, and transferred to a nylon membrane as described (L'Hernault and Arduengo 1992). The resulting blot was hybridized to a 779 bp radiolabeled genomic PCR product (Figure 4), which was amplified using the primer pair AC3-fwd3/AC3-rev3 (Table 2).
Total RNA was prepared from fem-3(q23gf) masculinized hermaphrodites (Barton et al. 1987) using the QIAGEN RNeasy kit (QIAGEN, Valencia, CA). A cDNA library was made from this RNA using a Clontech SMART II PCR cDNA Synthesis kit (BD Biosciences Clontech, Palo Alto, CA). The 5′ end of the spe-10 gene was amplified from the cDNA library by a nested PCR using the Clontech SMART II primer and two spe-10 gene-specific reverse primers, AC3-rev6 and AC3-rev7 (Table 2). The 3′ end of the spe-10 gene was amplified by a nested PCR reaction using the Clontech CDS primer and two spe-10 gene-specific forward primers, AC3-fwd10 and AC3-fwd11, that span exons 1-2 and exons 2-3, respectively (Table 2).
Some DNA sequencing was performed in-house on a BaseStation (MJ Research, Waltham, MA), while most was performed at either Iowa State University or the University of Michigan using standard ABI (Perkin-Elmer, Foster City, CA) automated fluorescent sequencing methods for polymerase chain reaction (PCR) products. Genomic DNA was prepared from picked spe-10 homozygous mutants and used to PCR-amplify the chromosome V AC3.10 gene (primer pairs AC3-fwd3/AC3-rev3 and AC3-fwd4/AC3-rev4; Table 2). The PCR products from four independent reactions for each genotype were pooled, purified using either phenol-chloroform extraction or the Qiaquick PCR purification kit (QIAGEN), and sequenced in order to identify the mutant lesions. Sequence traces from the spe-10 mutants were compared with the corresponding control sequence traces from N2 and also to the sequence available from the Sanger Centre (Hinxton, England). The plasmids pSL43 and pSL44 were sequenced on one strand to confirm that they were error-free.
Rabbit preimmune sera were screened by immunoblot and immunofluorescence at a dilution of 1/100 to identify sera that did not react with C. elegans proteins. Peptides 361 (CEKNRYVVEKSTPEQLAQ-COOH) and 577 (CQQRNRFVRIE EEPSSTQSSQSSIQ-COOH), respectively, corresponded to residues 112–128 and 328–351 of the SPE-10 polypeptide sequence (Figure 5). Peptide sequences were selected on the basis of antigenic probability (Hopp and Woods 1983) and <50% sequence identity with other C. elegans proteins. The (underlined) N-terminal cysteine of these peptides, which is not found in the SPE-10 sequence, was used to conjugate them to keyhole limpet hemocynanin carrier protein. Each conjugated peptide was used to immunize two rabbits using standard protocols (Zymed Laboratories, South San Francisco, CA); only the polyclonal antiserum 577-1 proved specific and is referred to as SPE-10 antiserum throughout this article.
Worm sperm were prepared for immunohistochemistry by the freeze/crack methanol fixation method (Miller and Shakes 1995). The only substantial variation from these methods involved use of Colorfrost/Plus microscope slides (Fisher Scientific, Pittsburgh) to facilitate adhesion and mounting in ProLong Antifade (Molecular Probes, Eugene, OR). Affinity purification of SPE-10 antiserum was performed with the immunizing peptide coupled to SulfoLink gel (Pierce Biotechnology, Rockford, IL). Crude anti-SPE-10 serum was incubated with the gel slurry and then washed with PBS containing 1% Tween and 1 m NaCl. A two-step elution of bound antibodies was performed, first in 3 m KSCN pH 7.0 and then in 0.1 m glycine pH 3.0, which was immediately neutralized with 1 m Tris-Cl, pH 8.0. The eluted fractions were separately concentrated to a final volume of 1 ml using CentriPrep columns (Millipore, Billerica, MA). The 0.1 m glycine affinity purified 577-1 antiserum was diluted to a final concentration of 50 μg/ml for all immunofluorescence experiments described in this article. A horseradish peroxidase (HRP)-conjugated goat anti- rabbit secondary antibody (Molecular Probes) was used at a final dilution of 50 μg/ml. Visualization was by 10 min of tyramine signal amplification (Bobrow et al. 1989; Chao et al. 1996) with Alexa Fluor 488 conjugated to tyramine (Molecular Probes). Mouse monoclonal antibody 1CB4 (Okamoto and Thomson 1985; Arduengo et al. 1998) was used at 1:2000 to detect the fibrous body-membranous organelle (FB–MO) complexes and was visualized using Texas Red-conjugated, affinity purified goat anti-mouse IgG secondary antibody (Fab1)2 fragment at a final concentration of 0.75 μg/ml (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were examined through appropriate filters under an Olympus BX60 microscope, and images were collected and analyzed as described above (see Phenotypic analysis).
A spe-10 cDNA was amplified by reverse transcription PCR with primers that added in-frame Eam1104 I restriction sites 5′ to the start ATG and 3′ to the last codon. This product was subcloned into the Eam1104 I sites of the pDual GC vector using the Seamless Cloning Kit (Stratagene) to create pEjG27, which was sequenced and confirmed to be error free. pEjG27 encodes the SPE-10 protein fused to a 6X His tag and a Myc epitope at its C-terminal end, which adds 6864 Da to the SPE-10 molecular weight. Recombinant BL21 (DE3) Codon Plus RIPL E. coli (Stratagene) were induced with 1 mm IPTG at 25° and lysed in a French pressure cell. Urea (6 M) was added to the bacterial lysate, and the 6X His tagged SPE-10 was purified using a His·Bind Column and Quick Buffer kit (Novagen, EMD Biosciences, Darmstadt, Germany). Whole worm lysates of fem-1(hc17 lf), fem-3(q23 gf), and him-8(e1489)IV; spe-10(ok1149)V were prepared by sonication (Hannak et al. 2002). Lanes on Tris-glycine mini gels (10% Life gels; Life Therapeutics, Clarkston, GA) were loaded with either a worm lysate (∼1,100 worms/lane) or ∼2 μg of purified SPE-10 recombinant protein and subjected to electrophoresis. Gels were transferred overnight to an Immun-Blot PVDF Membrane (Bio-Rad, Hercules, CA) and blocked with 5% non-fat dry milk. The Western blot was probed with SPE-10 577-1 KSCN affinity purified primary antibody (600 μg/ml) followed by a goat anti-rabbit IgG HRP conjugated secondary antibody at 1:5000 (Bio-Rad). ECL Plus (Amersham Biosciences, Piscataway, NJ) and BioMax MS Film (Kodak, Rochester, NY) were used for signal detection.
Genetic and phenotypic analyses of spe-10:
The spe-10 gene is defined by seven alleles including spe-10(hc104), for which the phenotype was characterized previously (Shakes and Ward 1989). Three new spe-10 alleles, spe-10(eb64), spe-10(eb105), and spe-10(eb106), were isolated in an ENU-induced F1 noncomplementation screen and the forward mutation rate for these mutations was ∼3 × 10−4. Two new spe-10 alleles, spe-10(eb112) and spe-10(eb118), were isolated in an EMS-induced noncomplementation screen and the forward mutation rate for these mutations was ∼4 × 10−4. The spe-10(ok1149) deletion mutation was recovered by the C. elegans Knockout Consortium after UV/TMP mutagenesis, and it proved to be a spe-10 molecular null mutation, as described below. All seven spe-10 mutants are strictly recessive for a self-sterile phenotype. Self-sterility, with no additional phenotypic features, is also observed when spe-10(eb112), spe-10(eb64), or spe-10(ok1149) is placed in trans to the noncomplementing deficiency sDf35 (Table 1).
All spe-10 mutants exhibit some degree of temperature sensitivity for their self-sterile phenotype. When grown at 25°, homozygous spe-10(eb64) or spe-10(ok1149) hermaphrodites did not produce any self-progeny, and all other spe-10 mutants produce very few self-progeny. At 16°, a group of three spe-10 mutants (hc104, eb64, or ok1149) are slightly self-fertile, producing 9–15 progeny (Table 1). The remaining four spe-10 mutants have a more dramatic temperature-sensitive Spe phenotype. At the 25° restrictive temperature, eb105, eb106, eb112, and eb118 produce less than four self-progeny per hermaphrodite, which is ∼2% of wild-type self-fertility, but at 16° each produces at least 50 self-progeny. The time period during development when spe-10(eb105) is temperature sensitive for fertility (temperature sensitive period; TSP) was determined by growing mutant hermaphrodites at 16° (the permissive temperature) or 25° (the restrictive temperature), shifting worms of various ages to the other temperature, and counting the total number of self-progeny produced by these hermaphrodites (Figure 2). The TSP for the sterile phenotype is between 30 and 45 hr of postembryonic development, which is during the L4 larval period when spermatogenesis occurs in the wild-type hermaphrodite (Hirsh et al. 1976). Two other genes known to play spermatogenesis-specific roles, fer-1 (Ward and Miwa 1978) and spe-9 (L'Hernault et al. 1988), each have a TSP that is very similar to spe-10.
Prior to our cloning of spe-10, light and electron microscopic study of spe-10(hc104) spermatogenesis revealed a spectrum of cytological defects in spe-10(hc104) mutants (Shakes and Ward 1989). We have re-examined the cytology of live cells isolated from spe-10(hc104) and six new spe-10 mutants (Figure 3). Wild-type spermatids bud from the residual body (rb) in a symmetrical fashion (Figure 3A), where each spermatid has a centrally located nucleus (in blue). In contrast, spe-10 spermatids bud from the rb in a more randomized fashion that has varying degrees of severity. Sometimes, well-formed spermatids that each contain a nucleus (in blue) are observed, as shown for the spe-10(ok1149) null mutant (Figure 3E). Other times, nuclei fail to leave the residual body and only two or three budding spermatids are observed, as shown for spe-10(ok1149) (Figure 3D) and spe-10(eb118) (Figure 3I). Prior electron microscopic observations of spe-10(hc104) noted a severe disturbance of FB–MO morphogenesis, and one consequence was that FBs remain in the residual body where they can bud as anucleate cytoplasts (Shakes and Ward 1989). Cytologically similar buds are observed on spe-10(ok1149) mutant residual bodies (arrowheads in Figure 3D), as well as those of other spe-10 mutants (not shown). All spe-10 mutants form spermatids; however, these spermatids tend to be smaller than wild type and they usually contain nuclei that are off center (see [ symbol in Figure 3E, G–I), as noted previously for spe-10(hc104) (Shakes and Ward 1989). A previously unreported defect is that spe-10 mutant spermatids (all alleles) can contain multiple nuclei as shown for spe-10(hc104) (* in Figure 3G) and spe-10(eb64) (Figure 3H). Mutant spe-10 spermatids and spermatozoa with two nuclei tend to be larger than those with a single nucleus.
Molecular cloning and identification of the of spe-10 gene:
spe-10 maps between unc-42 V and sma-1 V (Shakes and Ward 1989), which are cloned and located at positions +2.17 V (Baran et al. 1999) and +3.49 V (McKeown et al. 1998), respectively. spe-10 is located to the right of mDf1 and to the left of ctDf1 and fails to complement sDf35 (Figure 4A) (Shakes and Ward 1989). The physical distance between unc-42 and sma-1 is approximately 2.14 Mb of DNA (Coulson et al. 1995). Germline transgenic strains (Mello and Fire 1995) were created by injecting a series of overlapping YACs located within this interval. Phenotypic rescue of spe-10(hc104) hermaphrodite self-sterility occurred in worms transgenic for YAC Y37D6 (ebEx267), which contains ∼350 kb of DNA (Figure 4A). Cosmids within the Y37D6 region were then tested, and AC3 was found to include spe-10. Various restriction fragments derived from cosmid AC3 were then tested for phenotypic rescue of spe-10, initially using a variety of different mutants. Transgenes containing either of two restriction fragments, a 21,307-bp SacII/XhoI fragment (ebEx316; Figure 4) or a 3,332-bp SalI/SacI fragment (ebEx423; Figure 4), were found to restore self-fertility to spe-10 mutants and their effect on self-fertility was analyzed quantitatively in the spe-10(ok1149) null mutant (Table 1). ebEx316; spe-10(ok1149) was found to allow partial rescue in a temperature-sensitive manner, giving a brood of ∼130 (20°) and ∼40 (25°) (Table 1). We next examined the ebEx423 simple repeat transgene that contains a SalI/SacI epitope-tagged genomic restriction fragment called pSL43 (Table 1). AC3.10 was the only complete predicted gene contained within this SalI/SacI fragment (Figure 4A). Unlike ebEx316, ebEx423 bearing spe-10(ok1149) hermaphrodites grown at 20° had a self-brood size that was similar to wild-type N2 but had a small brood at 25°. In principle, this defect in 25° phenotypic rescue could have been due to either the epitope tag or something associated with rescue using a simple repeat transgene. We addressed these issues by using pSL44 to make the ebEx496 complex array (Kelly et al. 1997) that allowed spe-10(ok1149) to produce near wild-type sized self-broods at both 20° and 25°. These data indicate that the AC3.10 predicted gene is spe-10 and that the placement of a small HA epitope at the SPE-10 C-terminal end does not unduly interfere with its ability to function.
Many genes associated with a Spe phenotype encode a transcript that is specifically expressed during spermatogenesis (reviewed by L'Hernault 1997, 2005). Northern blots hybridized to a genomic PCR product that included the 5′ end of AC3.10 (Figure 4A) identified a ∼1.2 bp transcript in him-5(e1490) males (Hodgkin et al. 1979) and fem-3(q23 gf) masculinized hermaphrodites (Barton et al. 1987) that was not present in fem-1(hc17) feminized worms (Doniach and Hodgkin 1984) (Figure 4B). Both him-5 males and fem-3(q23) hermaprodites have a germ line that produces only sperm while fem-1(hc17) hermphrodites have a germ line that produces only oocytes.
spe-10 encodes a DHHC–CRD zinc-finger protein:
The ORF AC3.10, which encodes a 351 aa protein, was predicted by Genefinder (C. Wilson, L. Hilyer and P. Green, unpublished; see WormBase, http://www.wormbase.org). This predicted ORF was confirmed by cDNA sequence, and an additional 5′ noncoding exon was discovered. The predicted SPE-10 protein has a calculated mass of 41 kD, four predicted transmembrane domains (TMs) (Kyte and Doolittle 1982), and one potential N-glycosylation site (Marshall 1972) (Figures 5 and 6A). The most notable motif in SPE-10 is a DHHC–CRD zinc-finger (Bohm et al. 1997; Putilina et al. 1999) found between predicted TM2 and TM3 (Figures 5 and 6B).
The DHHC–CRD proteins (Pfam accession number: PF01529) are a diverse family of at least 346 eukaryotic proteins with a predicted zinc-finger motif that is defined by the spacing and nature of the zinc-coordinating residues. These proteins can have 0–11 TM domains and 306 of 346 proteins, including SPE-10, match the consensus motif C-X2-C-X9-H-C-X2-C-X2-C-X4-D-H-H-C-X5-C (Figure 6B; Bohm et al. 1997; Putilina et al. 1999). Loss-of-function phenotypes are known for four of the genes that encode DHHC–CRD proteins that match this consensus: ERF2 from yeast (Bartels et al. 1999), GODZ from mouse (Figure 6C; Keller et al. 2004), SPE-10 (this work; Shakes and Ward 1989), and SPE-21 (W. C. Lindsey and S. W. L'Hernault, unpublished results).
BLAST searches (McGinnis and Madden 2004) reveal that SPE-10 is most closely related to a diverse group of 38 DHHC–CRD proteins found in animals, including both soluble and TM proteins (not shown). A combination of Pfam searches (Bateman et al. 2004), SMART searches (Schultz et al. 1998; Letunic et al. 2004), hydropathy plots (Kyte and Doolittle 1982), and visual inspection found that 14/38 members of this group are predicted four-pass integral membrane proteins with the DHHC—CRD domain just before TM domain 3. All 14 proteins lack a cleavable N-terminal signal sequence or any other Pfam A domain (Bateman et al. 2004), which are frequently found in other DHHC–CRD proteins, and the 10 closest homologs were analyzed in detail (Figure 6C). These 10 DHHC–CRD proteins are 49% identical and 63% similar in a 49 aa region that includes the zinc-finger domain. These ten proteins also have significant homology in the region C terminal to TM domain 4, including a consensus Nx(S/T) glycosylation site (underlined NxT in Figure 6D; Marshall 1972).
Sequences of spe-10 mutant alleles:
AC3.10 and surrounding DNA were sequenced to determine the molecular nature of the mutations in this gene (Figures 5 and 6A). ok1149 is a deletion that removes 1,582 bp of DNA, and it affects two predicted open reading frames (Figure 4A). The deleted region spans the last 134 bp of 3′coding sequence of AC3.4 all the way to AC3.10 (spe-10) exon 4, including the spe-10 transcription start (Figures 4A and 5A). The ok1149 deletion is missing the coding information for the first 269 of the 351 SPE-10 amino acids, including the entire conserved DHHC–CRD region. spe-10(hc104) is a C-to-T transition at position 491 that causes a nonsense mutation converting arginine codon 115 (CGA) to a premature opal stop codon (TGA) and should create a truncated polypeptide chain. The spe-10(eb112) mutant contains a G-to-A transition mutation at position 90 in exon 2 that introduces a new start methionine codon 5′ to the wild-type spe-10 translation start codon. When the new spe-10(eb112) associated methionine codon is used as the start codon, the reading frame would be +1 relative to wild type and a novel 19 amino acid peptide would be encoded before translation terminated at the first in-frame stop (ochre) codon (Figure 5B). The last four mutants each contain missense mutations. spe-10(eb64) is a C-to-T transition at position 710 converting histidine codon 169 (CAT) to a tyrosine codon (TAT). This histidine is conserved in at least 306 DHHC–CRD predicted proteins (underlined in Figure 6, B and C). spe-10(eb105) is a T-to-A transversion at position 870 converting valine codon 222 (GTT) to an aspartic acid codon (GAT) between the predicted third and fourth transmembrane domains (Figure 6A). spe-10(eb106) is a T-to-A transversion at position 191 converting valine codon 31 (GTA) to a glutamatic acid codon (GAA) within the first predicted transmembrane domain (Figure 6A). spe-10(eb118) is a G-to-A transition at position 1110 converting glycine codon 302 (GGA) to a glutamic acid codon (GAA). This missense mutation is located C terminal to the predicted fourth transmembrane domain, and the glycine residue is conserved in a region where SPE-10 shows significant homology to the other DHHC–CRDs (underlined G, Figure 6D).
SPE-10 protein expression:
Antiserum 577-1 was raised to a synthetic peptide that corresponded to the C-terminal end of the SPE-10 protein (Figure 5). Full length 6X His-tagged recombinant SPE-10 was affinity purified from a bacterial lysate and was recognized by antiserum 577-1 (arrow in Figure 7, lane A). This fusion protein and all of its proteolytic products are also recognized by a MYC antiserum because it is tagged on its C-terminal end with this epitope (not shown). The 577-1 antiserum appears to be spermatogenesis-specific: it recognizes a protein of the predicted size in fem-3(q23) hermaphrodites that are somatically female but produce only sperm, but not in fem-1(hc17) hermaphrodites that produce only oocytes and no sperm or a spe-10(ok1149); him-8(e1489) population that was ∼37% males (Figure 7). The upper band for fem-3(q23) (lane C) is the expected molecular weight for wild-type SPE-10 while the lower band is presumably a SPE-10 proteolytic product.
SPE-10 localizes to the FB–MO:
Immunofluorescence was used to examine spermatocytes and spermatids from both (wild-type-like) him-8 control and him-8; spe-10(ok1149) male worms (Figure 8). Sperm were stained with both the 1CB4 mouse monoclonal antibody, which specifically recognizes FB–MOs in sperm (Okamoto and Thomson 1985; Arduengo et al. 1998), and the 577-1 SPE-10 antiserum. The control him-8 sperm have an anti-SPE-10 immunofluorescent signal that localizes specifically to budding and budded spermatids (Figure 8, C and G). This punctate signal was remarkably similar to that observed for the 1CB4 antibody (Figure 8, B and F) and the two signals largely, although not completely, overlapped (Figure 8, D and H). In contrast, him-8 spe-10(ok1149) sperm contained the antigen recognized by 1CB4 (Figure 8, J and N), but did not have a specific signal when stained with SPE-10 antiserum (Figure 8, K and O). This staining pattern indicates that spe-10(ok1149) spermatids contain MOs, as shown previously by EM (Shakes and Ward 1989), but that they lack SPE-10 protein, consistent with the interpretation that this mutant is a protein null. There is a diffuse SPE-10 antiserum background (arrow, Figure 8, G and O) observed in the rb, but this signal is present in both the him-8 control (Figure 8G) and him-8; spe-10(ok1149) (Figure 8O). These data indicate that SPE-10 mostly localizes within FB–MOs during spermatid formation.
The spe-10 gene encodes a novel, predicted four-pass integral membrane protein that contains a highly conserved DHHC–CRD motif (Bohm et al. 1997; Putilina et al. 1999). If a potential glycosylation site following TM4 is utilized, then the N-terminal region, the DHHC–CRD zinc-finger, and the C-terminal region should all face the lumen of the MO (Figure 6A). This orientation would allow the N-linked glycans to face the exterior of the cell surface when the MOs fuse to the plasma membrane. Northern hybridizations comparing oogenesis-specific and spermatogenesis-specific transcripts indicate that the spe-10 mRNA is found only in worms that are actively engaged in spermatogenesis (Figure 4B). SPE-10 localizes within the lysosome-like FB–MOs and segregates to spermatids as they bud from the residual body during C. elegans spermatogenesis (Figure 8). These results suggest that a lack of wild-type SPE-10 in the FB–MOs of spe-10 mutants probably causes the previously described sperm ultrastructural defects and self-sterile phenotype (Shakes and Ward 1989).
The spe-10(ok1149) mutant lacks most of the spe-10 gene and has no detectable signal when stained with a SPE-10 antiserum. These data indicate that spe-10(ok1149) mutants are null for SPE-10 function. Surprisingly, spe-10(ok1149), like all other spe-10 mutants (Table 1), is slightly temperature sensitive. The spe-10(hc104) opal nonsense and spe-10(eb64) missense mutants also have very weak temperature sensitivity. Both of these mutants are probably strong loss-of-function or null mutants because spe-10(hc104) should truncate SPE-10 synthesis N terminal to the DHHC-CRD domain and spe-10(eb64) changes an amino acid within the DHHC–CRD domain that is conserved in all homologs. The spe-10(eb112) mutation introduces a methionine codon 5′ to the wild-type start methionine (Figure 5). While this mutant only occasionally produces more than one progeny when propagated at 25°, it produces a brood that is ∼20% of wild type when it is propagated at 16°. Usually, eukaryotic translational initiation occurs at the first methionine codon as the ribosome scans from the 5′ end of the mRNA, but codon context also plays a role (reviewed by Kozak 2002). The context around the natural start methionine codon of spe-10, ACCAAUG (Figure 5A), is a poor match to the C. elegans consensus, which is AAAAUG (reviewed by Blumenthal and Steward 1997). In contrast, the sequence 5′ to the mutationally introduced methionine codon in spe-10(eb112) exactly matches the C. elegans start codon consensus (Figure 5B). It is possible that utilization of the wild-type methionine codon (despite its poor context) and not the first methionine codon is more efficient when spe-10(eb112) mutant hermaphrodites are grown at 16° rather than at 25°; such temperature-dependent effects on translation from alternative start codons have been noted for other genes (e.g., Liu et al. 1997). It is also possible that the spe-10(eb112) mutation affects the stability of spe-10-encoded mRNA. Transcripts that initiate translation at the spe-10(eb112) associated methionine codon are in a reading frame that would include a stop codon near the 5′ end of the mRNA (Figure 5B). The spe-10(eb112) encoded mRNA is a potential target for SMG-mediated degradation because the SMG surveillance system targets mRNAs with premature stop codons (reviewed by Anderson and Kimble 1997).
Examination of three spe-10 mutant alleles (ok1149, eb64 and eb112) in trans to the noncomplementing deficiency sDf35 (Table 1) showed that penetrance for the self-sterility phenotype is not significantly changed and no additional phenotypic defects are observed. These data suggest that SPE-10 function is confined to the male (spermatogenic) germ line, consistent with the observed expression pattern for spe-10. It further suggests that the spe-10(ok1149) spermatogenesis phenotype is not obviously affected by partial deletion of AC3.4, in addition to AC3.10 (spe-10; Figure 4A).
spe-10(eb105), spe-10(eb106) and spe-10(eb118) missense mutants all produce reasonably large broods when grown at the 16° permissive growth temperature but are nearly self-sterile when grown at 25°. The spe-10(eb105) mutant causes a V-to-D change at position 222. This mutation occurs between TM domains 3 and 4 in a residue conserved only in the C. briggsae ortholog, and the function of this region is presently not clear. The spe-10(eb106) missense mutant causes a V-to-E change at position 31 that reduces the hydrophobicity of the first predicted transmembrane domain. This valine at position 31 is predicted to be the most hydrophobic residue in the first transmembrane domain (Kyte and Doolittle 1982) and analysis of spe-10(eb106) encoded SPE-10 by SMART (Schultz et al. 1998; Letunic et al. 2004) suggests that TM domain 1 is shifted toward the C terminus so as to exclude the glutamic acid that is at position 31 in this mutant. Since 16°-grown spe-10(eb106) hermaphrodites produce self broods that are close to wild type in size and hermaphrodites grown at 25° produce only a few progeny (Table 1), perhaps such a shift occurs in a temperature-dependent manner. The spe-10(eb118) mutant has a G-to-E change at position 302. This nonconservative change occurs in a region C terminal to TM domain 4, and while this residue is conserved in the homologues of SPE-10 (underlined in Figure 6D), the function of this region is presently not clear.
The spe-10(eb64) missense mutation causes an H-to-Y change at position 169 within the DHHC–CRD zinc-finger motif (underlined in Figure 6, B and C), and this mutant synthesizes detectable SPE-10 protein that localizes within the FB–MOs (not shown). Since spe-10(eb64) is a probable null mutant when grown at 25°, this region of the DHHC–CRD motif is probably essential for proper SPE-10 function, even though the mutant protein appears to be within the correct compartment. The DHHC–CRD zinc-finger probably uses either this H or the adjacent C (#3 in Figure 6B) to coordinate Zn2+. The DHHC–CRD zinc-finger, like other more extensively characterized zinc-finger types (Bach 2000; Borden 2000; Wolfe et al. 2000; Gillooly et al. 2001), probably requires coordinated Zn2+ in order to function. Since tyrosine is not known to coordinate Zn2+, it is possible that SPE-10 synthesized by the spe-10(eb64) mutant is null because it fails to properly coordinate Zn2+. Alternatively, this region of SPE-10 might interact with an accessory protein, as is known to be the case for the DHHC–CRD protein Erf2p (see below; Lobo et al. 2002) and the spe-10(eb64) mutation might abolish this interaction.
A growing body of data suggests that proteins with a DHHC–CRD are palmitoyl transferases that catalyze linkage of the palmitate lipid to cysteine residues on substrate proteins. This was first shown for two S. cerevisiae proteins, Akr1p (Roth et al. 2002) and Erf2p (Lobo et al. 2002); only the latter matches the consensus in Figure 6B. Highly purified Akr1p, which has a variant DHHC motif that is DHYC, was shown to palmitoylate the yeast casein kinase Yck2p (Roth et al. 2002). Site directed mutations in which the AAYC or DHYA replaced the DHYC motif of Akr1p abolished its ability to catalytically transfer palmitate to Yck2p. Erf2p is required for the catalytic transfer of palmitate to Ras2p, and two site-directed mutations in the DHHC region each abolish Ras2p palmitoylation in an in vitro assay system. Mutating the fourth Cys in the Erf2 consensus (Figure 6B) abolished interaction with a required interacting protein (ERF4p), while mutating the His next to the sixth Asp still had normal ERF4p interaction, but presumably interfered directly with catalytic activity (Lobo et al. 2002). These data show that the DHHC–CRD region is important in catalytic palmitoylation.
The typical mammalian genome encodes 23 DHHC–CRD proteins (Fukata et al. 2004) and several have been connected to palmitoylation of target proteins. HIP14 is a brain-expressed DHHC protein that was identified in a yeast two-hybrid assay as interacting with huntingtin (htt), which is a ubiquitously expressed protein implicated in Huntington's disease. HIP14 can functionally replace AKR1 in yeast mutants (Singaraja et al. 2002) and, in addition to htt, palmitoylates at least four other vesicular trafficking proteins found in the human brain (Huang et al. 2004). Four mouse DHHC–CRD proteins have been shown to act as palmitoyl transferases on PSD-95, which is essential for regulation of both synaptic plasticity and AMPA receptors (Fukata et al. 2004). One of these four DHHC–CRD proteins, DHHC-3 (Fukata et al. 2004) or GODZ (Uemura et al. 2002), was also independently identified in a two-hybrid screen for proteins that interact with the γ2 subunit of the GABA receptor (Keller et al. 2004). This protein is found in many tissues where it is localized to the Golgi complex. GODZ (#11 in Figure 6C) is also very similar to the DHHC–CRD subfamily that includes SPE-10. Both SPE-10 and GODZ are four-pass integral membrane proteins, and GODZ has a topology that is similar to that shown for SPE-10 in Figure 6A (Keller et al. 2004). Palmitoylation has been known to be an important regulatory event in vesicular trafficking pathways and a variety of intracellular regulatory proteins (reviewed by Smotrys and Linder 2004), but the enzymes catalyzing this lipidation have only recently been discovered. It now seems clear that the DHHC–CRD proteins are responsible for many of the palmitoylation events that occur in eukaryotic cells.
BLAST searches (McGinnis and Madden 2004) with the consensus motif shown in Figure 6B reveal that the C. elegans genome encodes 21 DHHC–CRD proteins from 17 genes. All but one of these genes has been subject to RNA interference (RNAi) experiments (Fire et al. 1998; Timmons and Fire 1998) and in all cases (including spe-10) RNAi-treated worms have no obvious phenotype (see WormBase, http://www.wormbase.org). Consequently, it is possible that conventional genetic analyses will be required to learn the loss-of-function phenotypes associated with C. elegans DHHC–CRD genes. At this point, SPE-10 is the only C. elegans DHHC–CRD gene to be subject to detailed genetic and phenotypic analysis. Our analysis indicates that SPE-10 is an integral membrane protein mostly located in the MO. SPE-10 is likely to function as a modulator of protein-protein interactions by palmityolating one or more substrates required for proper interaction of the MO with the FB. A mutant SPE-10 might not allow stable association between the FB and MO, abolishing transport of the FB into the spermatid. Future work will concentrate on identifying the targets of palmitoylation by SPE-10 and defining how this protein and any interacting partners participate in the membrane morphogenesis required for FB–MO function during C. elegans spermatogenesis.
We thank Alan Coulson and Andrew Fire for providing C. elegans DNA clones, Cornelia Bargmann for the pGETP1 plasmid, and John Logsdon for help in analyzing the DHHC–CRD protein family. The Caenorhabditis Genetics Center provided some nematode strains and is funded by the National Institute of Health National Center for Research Resources (NCRR). spe-10(ok1149) was provided by the C. elegans Gene Knockout Project at OMRF, which is part of the International C. elegans Gene Knockout Consortium, see http://www.mutantfactory.ouhsc.edu/. This work was supported by U. S. Public Health Service Grant GM40697 to S. W. L., funds from Emory College, and funds from an Emory University Research Committee award.
↵1 Present address: Waksman Institute, Rutgers University, Piscataway, NJ 08854.
Communicating editor: B. J. Meyer
- Received June 27, 2005.
- Accepted August 17, 2005.
- Copyright © 2006 by the Genetics Society of America