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Section of Molecular and Cellular Biology,4
Department of Cell Biology and Human Anatomy, School of Medicine,5 University of California, Davis, Davis, California 95616
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, gamete biology, sperm, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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jw7g/). Relevant to this study, four spermatogenic cell-specific ADAMs, fertilin alpha a, fertilin alpha b, fertilin beta, and cyritestin (products of the Adam1a, Adam1b, Adam2, and Adam3 genes, respectively), have been previously analyzed. Fertilin alpha a is distinctive in this group because it is not expressed on mature sperm, but only in spermatogenic cells. Fertilin alpha a (also known as ADAM1A) and fertilin beta (ADAM2) form a heterodimer that remains intracellular, apparently as a resident of the endoplasmic reticulum (ER) [2]. Fertilin beta also forms a heterodimer with fertilin alpha b (ADAM1B) that is expressed on the surface of mature sperm [2]. The in vivo functions of fertilin alpha a, fertilin beta, and cyritestin (also known as ADAM3) have been evaluated by gene deletion. In each case, homozygous males are infertile and sperm exhibit little or no binding to the zona pellucida of eggs in vitro [3–6]. Additionally, sperm from Adam1a-null or Adam2-null animals are unable to enter the oviduct. These defects block fertilization, demonstrating the functional importance of these proteins in reproduction. When these knockouts were studied further, a surprising molecular-level phenotype was discovered. In addition to the genetically deleted protein, one or more other ADAM proteins were found at reduced levels on mature sperm (Table 1). Fertilin alpha b is slightly reduced on Adam1a- and Adam3-knockout cells, but is completely lost from Adam2 knockouts. When Adam3 is deleted, fertilin beta is reduced to 75% of wild-type levels on sperm. The Adam1a knockout also exhibits a similarly mild loss of fertilin beta [4]. Cyritestin expression is much more drastically affected by the deletion of either Adam2 or Adam1a; on mature sperm from either knockout, cyritestin is present at about 10% of wild-type levels [3, 4]. Thus, the loss of cyritestin in the Adam2 knockout line is a principal focus of this study.
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The loss of fertilin beta in Adam3 knockouts and cyritestin in Adam2 knockouts occurs despite normal synthesis of these proteins during spermatogenesis [3, 4]. In this study, we investigated how posttranslational loss of these proteins might occur. We examined the processing and trafficking of fertilin beta and cyritestin in wild-type and Adam2- and Adam3-knockout mice. Wild-type processing consists of several steps that occur at specific stages of spermatogenesis. We found that processing in the knockouts was largely unchanged, but showed some subtle differences. The most dramatic changes we found in the knockouts occurred during the formation of testicular sperm, when most of the untargeted ADAM protein is lost. We also discovered that during the formation of testicular sperm, some proteins become sequestered in a Triton X-100 insoluble compartment, indicating that a specific sorting or retention pathway may exist. Our findings indicate that testicular sperm are a key developmental step in the regulation of surface expression and retention of ADAMs, and possibly other surface proteins. Our study suggests that members of the ADAM family may cooperate in the secretory pathway in wild-type testis to bring about normal surface expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ten- to 12-wk-old C57Bl/6 mice were purchased from Charles River Laboratories. Adam2–/– (fertilin beta-null) and Adam3–/– (cyritestin-null) mice were previously generated [3, 5]. All animal investigations were approved by the University of California, Davis Animal Care and Use Committee. Testes were removed, roughly chopped with dissection scissors, and cells were separated by four passes with a Dounce homogenizer. Testicular cells, which include spermatocytes and spermatids, and testicular sperm were isolated from the homogenate on a 52% Percoll/Mg++-Hepes gradient (Amersham Biosciences), washed, and resuspended in PBS [7]. The Percoll gradient yields a population of testicular germ cells close to the top of the gradient, while testicular sperm migrate as a band near the bottom of the gradient. For epididymal sperm isolations, the caput epididymis or the cauda epididymis and vas deferens were removed from adult male mice and placed into 37°C PBS. Mature sperm samples were taken from the cauda epididymis. This region contains a largely uniform population of cells that are fertilization-competent. Tissues were cut into small pieces using dissection scissors and sperm were allowed to swim out for 15 min at 37°C. Tissue fragments were removed and sperm were washed three times by brief centrifugation in PBS at room temperature before use. Cells were either resuspended directly into sample buffer containing 3% SDS (1x) and heated at 95°C for 5 min or were lysed in PBS containing 1% Triton X-100 and a protease inhibitor cocktail (Sigma) on ice for 1 h. Supernatant and pellet from Triton X-100 lysis were separated by centrifugation at 16 000 x g for 10 min. Pellets were directly resuspended in 1x sample buffer as above and supernatants were mixed with an equal amount of 2x sample buffer. Both were heated at 95°C for 5 min to solubilize protein.
Subcellular Fractionation
Sperm were removed from male mice as described above and purified away from contaminating cells and debris by centrifugation for 20 min at 400 x g onto the interface of a 25%/40% Optiprep (Sigma)/M199 (Life Technologies) step gradient. Sperm were removed from the gradient, washed once in 3 ml of M199 for 10 min at 350 x g, and capacitated for 1 h in M199 (Life Technologies) supplemented with 450 µM sodium pyruvate (Life Technologies) and 100 U/ml penicillin-streptomycin (Life Technologies) at 37°C. To induce the acrosome reaction, sperm were incubated for 1 h with 25 µM A23187 (Sigma) at 37°C. The treated sperm suspensions were centrifuged at 2000 x g to separate sperm from the acrosomal contents and vesicles were released by the acrosome reaction. Sperm were washed once in PBS and the pellet was resuspended in 1x sample buffer. The supernatant from the 2000 x g spin was subjected to ultracentrifugation at 200 000 x g for 2 h. The supernatant was collected and concentrated in a 10 000 MWCO Centricon tube (Millipore) and designated the acrosomal contents fraction. The pellet of the ultracentrifuge spin was resuspended in 2 ml of PBS and ultracentrifuged again under the same conditions. The membrane pellet was resuspended in 1x sample buffer and heated at 95°C for 5 min to solubilize protein.
Immunoblotting
Samples were separated by SDS-PAGE on 8% or 10% Novex gels (Invitrogen) under reducing conditions (50 mM dithiothreitol [USB]), transferred to Immobilon-P PVDF membrane (Millipore) and blocked for 1 h at room temperature in 5% dry milk in TBS + 0.05% Tween 20 (Bio-Rad). Antibodies used for immunoblotting included 9D2.2 (mouse monoclonal antifertilin beta) and 7C1.2 (mouse monoclonal anticyritestin) (both from Chemicon), rabbit polyclonal anti-PH-20 [8], and rabbit polyclonal antitestase in 1 (ADAM24) antibody [9]. Blots were incubated with alkaline phosphatase-conjugated secondary antibodies against rabbit immunoglobulin G (IgG) or mouse IgG (Pierce) and detected with NBT/ BCIP (Pierce). Blots were quantified using ImageQuant. For each experiment to determine protein loss, an identical gel was blotted with antitestase in 1, which was used as a loading control. It has previously been shown that testase 1 levels do not change in Adam2 or Adam3 spermatogenic cells [3]. Three to nine replicates of these quantification experiments were performed and quantification is presented ± SEM.
Biotinylation of Cell Surface Proteins
Cells were isolated as described above and resuspended in PBS. Membrane-impermeable biotinylation reagent EZ link sulfo-NHS-LC-biotin (Pierce) (500 µg/ml) was incubated with 3 x 106 cells for 30 min at room temperature. To maximize biotinylation, an equivalent amount of biotinylation reagent was reintroduced into the sperm sample and the incubation was repeated. Control samples were incubated in the absence of biotinylation reagent. The biotinylation reaction was quenched for 30 min in 25 mM glycine. Sperm were centrifuged at 2000 x g for 5 min to pellet, washed three times in cold PBS, and resuspended in 1x sample buffer. The control and treated samples were separated by SDS-PAGE and analyzed by immunoblot for a shift in molecular weight.
Glycosidase Digestion
Cells were isolated and lysed in PBS + 1% Triton X-100 as described above. Peptide N-glycosidase F (PNGase F) digestion was performed under native conditions on 1% Triton X-100 lysates for 1 h at 37°C with 0.4 µl of enzyme (500 U/µl, New England Biolabs) per 1 x 106 cells. Cell samples were prepared for Endoglycosidase H (Endo H) digestion by the introduction of 2x denaturing buffer (1% SDS and 2% ß-mercaptoethanol) to an equal volume of lysate and incubation at 95°C for 10 min. Sodium citrate (pH 5.0) was added to a final concentration of 0.2 M along with Endo H (Roche) at a concentration of 160 mU/ml. The samples were incubated at 37°C for 1 h. For both PNGase F and Endo H digestion, the reaction was stopped by the addition of an equal volume of 2x sample buffer, followed by a 5 min incubation at 95°C.
Low SDS Nonreducing Electrophoresis
Testicular cells and testicular sperm were isolated as described above and solubilized in PBS + 1% NP-40, as described by Cho et al. [10]. Supernatants of the lysates were then mixed with sample buffer to a final concentration of 0.3% SDS. The samples were incubated at room temperature for 5 min and separated by nonreducing SDS-PAGE with a hand-cast 7.5% gel at 4°C. Proteins were transferred to polyvinylidene difluoride as described above and the membrane was soaked in 9 M ß-mercaptoethanol in 50 mM Tris-HCl pH 6.8, 2% SDS for 30 min in a 50°C water bath to denature proteins for immunoblotting. The blot was washed thoroughly in water before immunoblotting.
Removal of Cytoplasmic Droplets
Testicular sperm were isolated by Percoll gradient (as above) and 100 cells were counted to assess the percentage of cells with cytoplasmic droplets (average =49%). Half of the sample was reserved and half of the sample was resuspended in PBS and placed on top of a 0.25 M/1.0 M sucrose step gradient [11] and centrifuged at 1500 x g for 20 min. The pellet was resuspended in PBS and cytoplasmic droplets were counted (average =16% intact cytoplasmic droplets). Sample buffer was added to both cell populations to solubilize for SDS-PAGE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the protein loss that occurs in the Adam2-null and Adam3-null sperm, it was first necessary to determine the wild-type patterns of fertilin beta (protein product of the Adam2 gene) and cyritestin (protein product of the Adam3 gene) processing. We discovered that concomitant with the maturation of the spermatogenic cell, fertilin beta and cyritestin are processed in several distinct steps, the timing of which is precisely controlled. Protein processing may regulate protein activity at different stages of spermatogenesis.
Fertilin Beta
As has been observed previously, fertilin beta is present on mouse testicular cells as a full-length protein with a molecular mass of 100 kDa [5]. The 100-kDa protein is also the major form on testicular sperm; in addition, 12% ± 1.99% of fertilin beta is present as an 80-kDa intermediate (Fig. 1A, lane TS). Sperm from the caput epididymis have undergone a major processing event that removes approximately half of the protein mass, yielding a 45-kDa product (Fig. 1A, lane CT). Fertilin beta on cauda epididymal sperm migrates slightly faster, 42 kDa (Fig. 1A, lane CA). The small epididymal molecular mass change of fertilin beta (and cyritestin, see below) probably results from protein processing because the molecular masses of these proteins remain different following treatment with PNGase F to remove glycosylation (data not shown). Following the acrosome reaction, fertilin beta remains associated with the acrosome-reacted sperm and a subset of the protein is processed to a 27 kDa form (Fig. 1A, lane AR).
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Cyritestin
Cyritestin is detected as a full-length protein (110 kDa) on testicular cells [12] and testicular sperm (Fig. 1B, lanes TC and TS). Full-length cyritestin on testicular sperm exhibits some heterogeneity, but in contrast to fertilin beta, no processing intermediate is reproducibly observed (Fig. 1B). Processing of cyritestin in the epididymis is similar to the pattern observed for fertilin beta, with the major cleavage occurring by the caput epididymal stage and a second minor cleavage of 2 kDa detectable in the cauda epididymal sperm, generating protein products of 42 kDa and 40 kDa, respectively (Fig. 1B, lanes CT and CA). Cyritestin is released in the soluble acrosomal fraction following the acrosome reaction (Fig. 1B, lane AC), a result also recently reported by Kim et al. [13]. This finding raises the question of how a transmembrane protein such as cyritestin is released from the sperm. We detected a small molecular mass change in cyritestin, from 40 to 38 kDa following the acrosome reaction (Fig. 1B, compare lanes AR and AC). This change is consistent with the loss of the transmembrane domain as the result of a membrane-proximal cleavage of the ectodomain or may reflect other proteolytic modifications by released acrosomal proteases.
Reduction of Protein Levels in the Knockout Mice Occurs Primarily During Late Stages of Testicular Sperm Formation
Once a comprehensive picture of wild-type processing was established, knockout cells were examined to determine the timing of ADAM protein loss and whether any alterations in the processing patterns were present. Previous work has shown that when Adam2 is deleted, the level of cyritestin is dramatically reduced on mature sperm; also, when Adam3 is deleted, the level of fertilin beta is slightly reduced on mature sperm [3]. These two proteins are present at wild-type levels in testicular cells of both knockouts, indicative of normal levels of transcription and translation ([3] and present work). Thus, we asked whether the loss of these proteins occurred in the testis or epididymis by examining testicular sperm, the final spermatogenic stage in the testis. A 25% ± 3.12% reduction in the levels of fertilin beta on Adam3-null testicular sperm was observed when compared to wild-type levels, equivalent to the total loss observed on mature sperm (Fig. 2A). Additionally, in Adam3-null testicular sperm, we observed precocious processing of fertilin beta. Whereas in wild-type mice 12% ± 4.5% of fertilin beta is present in the 80-kDa form, in Adam3-knockout mice, 25% ± 5.2% of the remaining fertilin beta protein is now present as an 80-kDa intermediate (Fig. 2A). These findings demonstrate that loss of fertilin beta in Adam3-knockout mice occurs before the cells exit the testis.
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The loss of cyritestin protein in the Adam2-null males is much more severe. At the testicular sperm stage, cyritestin protein is reduced by 65% ± 7.08% compared to levels in wild-type testicular sperm (Fig. 2B). Additional cyritestin is lost during epididymal passage, resulting in a 90% ± 7.1% reduction from wild-type levels (Fig. 2B). However, no major alteration in the protein processing is observed. In summary, the protein loss in each knockout exhibits a unique timing. Fertilin beta is lost in one step in the testis, while cyritestin loss occurs in both the testis and the epididymis. This suggests that the mechanism of loss may be different in the two knockout mouse lines.
The Cytoplasmic Droplet Is Not Responsible for Epididymal Loss of Cyritestin
As noted above, additional cyritestin is lost from sperm in the epididymis of Adam2-knockout mice. We hypothesized that this small population of cyritestin might be contained in the cytoplasmic droplet, a cytoplasm-filled structure that remains associated with sperm after the bulk loss of cytoplasm. Cytoplasmic droplets contain remnants of intracellular organelles such as the ER and Golgi, and are shed from most sperm cells during epididymal transit [14]. To this end, we compared the protein loss in testicular sperm with the cytoplasmic droplets intact with testicular sperm from which the cytoplasmic droplets had been removed (Fig. 3). We found that removal of the cytoplasmic droplet does not recapitulate the epididymal loss of cyritestin, and thus some other mechanism may account for this loss.
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Trafficking of Fertilin Beta and Cyritestin to the Surface of Wild-Type Cells
We have shown that the majority of protein loss in both knockout lines occurs in the testis following normal levels of translation. Posttranslational protein loss can occur when transport of a protein through the secretory pathway is disrupted. We monitored this process for fertilin beta and cyritestin using two hallmarks of progress through the secretory pathway: acquisition of Endo H resistance and cell surface localization. Resistance to Endo H is indicative of glycosylation modifications conferred upon glycoproteins in the medial Golgi. We examined glycosidase sensitivity of fertilin beta and cyritestin in the testis. PNGase F treatment confirmed the presence of glycosylation on both proteins (Fig. 4, A and B). Most of the fertilin beta and most cyritestin is Endo H-sensitive in wild-type testicular cells, and the same pattern of sensitivity is observed for knockout cells (Fig. 4A). Therefore, at this developmental stage, most of the fertilin beta and cyritestin is localized in a pre-Golgi compartment, probably the ER. In contrast, on wild-type testicular sperm, both fertilin beta and cyritestin are Endo H-resistant (Fig. 4B). A similar (but not identical) pattern of Endo H resistance is observed on knockout testicular sperm. It should be noted that for both fertilin beta and cyritestin, the Endo H-resistant form of the protein does not comigrate with the control untreated protein, indicating that some glycosylations remain Endo H-sensitive (Fig. 4B).
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To test whether the proteins reach the cell surface in the knockouts, surface localization on testicular sperm was assayed by incubating intact cells with a nonpermeable biotinylation reagent. Cell surface proteins are accessible to modification by biotin, which results in a slightly slower migration on SDS-PAGE than the nonbiotinylated form. Both fertilin beta and cyritestin on wild-type testicular sperm are biotinylated, indicating surface localization (Fig. 5, WT). Experiments with a spermatogenic cell transmembrane protein, calmegin, did not show a molecular weight change (data not shown). On testicular sperm isolated from Adam3 knockout mice, biotinylated fertilin beta is also detected (Fig. 5, C –/–), indicating surface localization. In striking contrast, we did not observe biotinylated cyritestin on Adam2-knockout testicular sperm (Fig. 5, beta –/–), demonstrating low or no surface expression on these knockout sperm.
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Fertilin Beta and Cyritestin Are Present in Several High Molecular Weight Protein Complexes
Once the timing of loss was determined, we asked about the mechanism of loss. A straightforward explanation for protein loss in these knockouts is that the formation of a complex containing fertilin beta and cyritestin is essential for proper folding and trafficking of both components. We examined this question by using low-SDS, nonreducing conditions to monitor complexes containing either protein in the wild-type and knockout lines. The fertilin alpha a/ fertilin beta and fertilin alpha b/fertilin beta complexes are observed at 220 and 200 kDa [2, 10]. In the absence of cyritestin there is no change in these complexes on testicular cells (Fig. 6A). In wild-type testicular sperm, small amounts of the 220/200 kDa complexes are observed, but the major form of fertilin in these cells is 160 kDa, which is consistent in size with a complex of processed fertilin alpha b and full-length fertilin beta. In Adam3-null testicular sperm no major differences from the wild type are observed, indicating that fertilin beta and cyritestin do not form a complex. This conclusion is also supported by the cyritestin data. In testicular cells we were able to detect two cyritestin complexes at 246 and 221 kDa. Judging from the detection intensity, these complexes represent a very small percentage of the cyritestin population. This is the first evidence that cyritestin has partners, and while the identities of these proteins are unknown, neither is altered in the absence of fertilin beta. No cyritestin complexes are observed on wild-type or Adam2-knockout testicular sperm. In sum, these results suggest that the protein loss in the knockouts does not result from a failure to form a fertilin beta-cyritestin complex, and that the interaction between these proteins is likely of another kind.
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Fertilin Beta and Cyritestin Exhibit Transient Triton X-100 Insolubility at the Testicular Sperm Stage that Is Not Observed in Fertilin beta Null Cells
In the course of analyzing ADAM protein loss, we discovered that some plasma membrane proteins become transiently insoluble in Triton X-100 at the testicular sperm stage. A significant fraction of these proteins are not released from the testicular sperm cell by treatment with 1% Triton X-100 for 1 h at 4°C. This observation is most dramatic for cyritestin, in which most of the protein (70%) is insoluble at this stage. Cyritestin shows much higher solubility after Triton X-100 lysis in testicular cells earlier in spermatogenesis (only 28% insoluble) and also in cauda epididymal sperm (only 1% insoluble) (Figs. 7, 8A). This phenomenon was not universally observed among testicular sperm surface proteins. Three additional proteins were tested, and although all exhibited low insolubility in testicular cells and mature sperm, the degree of insolubility in testicular sperm was variable (Fig. 8A). Moderate insolubility was observed for fertilin beta (54% insoluble) and testase 1 (ADAM24) (47% insoluble), whereas PH-20 remains highly soluble in testicular sperm (22% insoluble). We tested whether this transient insolubility was maintained in the testicular sperm of knockout males. We detected no or very small alterations in the solubility patterns for the tested proteins in either knockout, with the exception of cyritestin in Adam2–/– cells (Figs. 7 and 8B). In Adam2-null testicular sperm, the remaining cyritestin retained the same relatively low level of insolubility as it has in testicular cells (30%).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The secondary loss of untargeted plasma membrane proteins in mouse knockouts has been reported for other proteins. Most often, the loss is due to cellular quality control mechanisms and occurs when the deleted gene encodes a protein that is part of a multimeric complex [15, 16]. The orphaned subunit(s) form an improperly assembled complex, which is detected by ER quality control elements and targeted to the proteasome through a process known as Endoplasmic Reticulum Associated Degradation (ERAD) [17]. The complete loss of fertilin alpha b on Adam2-null mature sperm can probably be attributed to this type of mechanism, as these two proteins form a heterodimer [10]. However, fertilin beta and cyritestin loss in the Adam3-null and Adam2-null cells, respectively, is not likely to occur in this way. No complex has been observed between these proteins, despite a major effort to this end ([3, 4] and present work).
Cyritestin loss from Adam2-null cells occurs in both the testis and epididymis. On both wild-type and Adam2-null testicular sperm, much of the cyritestin has passed through the medial Golgi (as judged by Endo H resistance), but on the mutant sperm, no cyritestin is detected on the cell surface. This suggests that in the absence of Adam2, cyritestin trafficking from the trans-Golgi to the plasma membrane is disrupted or that cyritestin is shed or degraded shortly after appearing at the cell surface. If cyritestin cannot proceed from the Golgi to the surface in Adam2-null sperm, loss may be due to retention in the residual body or proteolytic degradative processes. The similar extent of cyritestin loss (10% wild-type levels) in the Adam2-null and Adam1a-null sperm might be explained by a chaperone-like activity of the fertilin alpha a/fertilin beta heterodimer. The fertilin alpha a/fertilin beta heterodimer has been reported as an ER resident protein [4]; thus, it may be required for a critical step for cyritestin (e.g., correct folding) that occurs in the ER, but leads to post-Golgi quality control events if the ER step is defective.
This type of chaperoning activity has been noted in other cases in which knocking out one membrane protein leads to a posttranslational reduction in surface expression of another protein. This process is distinct from ER quality control because these proteins are not subunits of an obligate multimeric complex. One clear case is the Cd81 knockout, in which CD19 is made but very little (10%) reaches the surface [18]. CD19 in Cd81-null B cells is present at normal levels in the ER, but the Endo H-resistant fraction is reduced by 50%. Like cyritestin in the Adam2 knockout, CD19 surface expression may be reduced by retention in the secretory pathway or by subsequent proteolytic cleavage at the cell surface. Therefore, both fertilin beta-cyritestin and CD81-CD19 engage in a currently undefined nonreciprocal relationship that, when disrupted, leads to a loss of one protein in a post-ER compartment, resulting in a severe reduction of cell surface expression.
In our study we detected a Triton X-100 insoluble fraction on testicular sperm containing a subset of plasma membrane proteins. Segregation of sperm proteins into a Triton X-100 insoluble fraction has not previously been observed. Among the proteins that we studied, cyritestin shows the highest level of residence (70%) in this detergent-insoluble compartment. In Adam2-null testicular sperm, cyritestin was not properly segregated and only 28% of the protein was found in the insoluble fraction. The correlation of low cyritestin residence in the insoluble compartment and the sizeable reduction in total cyritestin (60%–80% loss) by the testicular sperm stage is consistent with the idea that the sorting into the insoluble compartment is required for cyritestin's perdurance on testicular sperm.
Triton X-100 insolubility is consistent with several mechanisms that could serve to sort proteins. Proteins in the late Golgi may be packaged into a distinct Triton X-100 insoluble microenvironment for delivery to the cell surface. Alternatively, the insoluble fraction may be formed after the membrane protein reaches the cell surface of testicular sperm. There, cytoplasmic tail association with an intracellular component could be responsible for detergent insolubility. If this intracellular component was restricted to a certain region of the cell, it could be used as a mechanism to localize or secure surface proteins into plasma membrane domains. The proteins we examined did not remain in the Triton X-100 insoluble fraction at the mature sperm stage. The transient nature of this subpopulation may indicate that insolubility might only be necessary to establish a domain, but not to maintain it.
In conclusion, we have analyzed a phenomenon previously described in studies of targeted deletions of sperm ADAMs. In addition to the loss of the deleted protein, one or more other ADAMs show reduced levels [3, 4]. This process is not restricted to ADAMs or sperm, because developing B cells, null for the tetraspanin Cd81, express 10-fold reduced levels of the signaling molecule CD19 on the cell surface [18]. We suggest that fertilin beta and CD81 have chaperone-like activity, perhaps revealing a cooperative nature of protein passage through the secretory pathway to bring about surface expression. This cooperation may be part of a posttranslational control system that regulates correct expression of membrane proteins during development and in specific adult tissue.
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FOOTNOTES |
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2 Correspondence. Fax: 530 752 7522; dgmyles{at}ucdavis.edu
3 Current address: Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Received: 15 February 2005.
First decision: 16 March 2005.
Accepted: 8 July 2005.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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