A Developmentally Regulated Chaperone Complex for the Endoplasmic Reticulum of Male Haploid Germ Cells

Abstract

Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide ⌬-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility. INTRODUCTIONProtein disulfide isomerase (PDI) catalyzes the formation, isomerization and reduction of disulfide bonds in the endoplasmic reticulum (ER). More than 17 mammalian PDI homologues have been identified all characterized by the presence of one or more thioredoxin folds (Ellgaard and Ruddock, 2005). PDI itself contains four of these thioredoxin domains: two with the thioredoxin redox active site CXXC (designated a-type domains) and two domains lacking the active site (designated b-type domains). The b-type domains lack redox activity, but have either a structural function or are involved in substrate recognition and binding (Klappa et al., 1998a). In PDI, these domains have an a-b-bЈ-aЈ organization. PDIp, ERp57, and PDILT share the same domain structure with PDI, but various other configurations of thioredoxin domains exist in other PDI family members, with most PDIs containing at least one a-type domain with an intact active site motif (Ellgaard and Ruddock, 2005).PDI homologues are not functionally equivalent, as shown by differences in their ability to complement a yeast strain deficient for PDI (Gunther et al., 1993;Kramer et al., 2001). Furthermore, the different PDI family members interact with discrete sets of substrates. PDI catalyzes in vitro redox reactions in a wide variety of substrates, including bovine pancreatic trypsin inhibitor (BPTI), insulin, and ribonuclease (Weissman and Kim, 1993;Zheng and Gilbert, 2001). PDI also displays chaperone activity toward nondisulfide bond-containing substrates (Cai et al., 1994) and forms the noncatalytic subunit in prolyl 4-hydroxylase (Koivu et al., 1987) and microsomal triglyceride transfer complex (Wetterau et al., 1990). ERp57 interacts with a specific set of glycosylated proteins that are recruited via its interaction with the lectins calnexin/calreticulin (Oliver et al., 1997;Jessop et al., 2007) and is a component of the major histoco...

Protein disulfide isomerase homolog PDILT is required for quality control of sperm membrane protein ADAM3 and male fertility

“…S1 A and B). PDILT was specifically detected in testis, consistent with previous observations in rat and human (23,24). In developing mouse testes, whereas PDIA3 (ERP57) was continuously detected throughout development, PDILT was not detectable until the mice became 3 wk old, indicating postmeiotic expression.…”

“…1 A and B), because CLGN-and CALR3-deficient mice had infertile phenotypes attributed to failures in ADAM1/ADAM2 heterodimerization and ADAM3 maturation, respectively (11). PDILT interacted with neither CANX nor CALR, but did bind to CLGN in wild-type mice, consistent with the data from rat (23). We further showed that PDILT interacted with CALR3 and that the signal was stronger than with CLGN (Fig.…”

“…In addition to ubiquitous PDIs including PDIA3, male germ cells also express a testis specific PDIlike protein, PDILT (22). Although PDILT does not have consensus CXXC motifs and is not a classical oxidoreductase, it does interact with CLGN and with model proteins in vitro, suggesting that it may have a role in the maturation of spermatid proteins and consequently, male infertility in vivo (23).…”

Calsperin Is a Testis-specific Chaperone Required for Sperm Fertility

“…Recently, a testis-specific homolog of protein-disulfide isomerase (PDILT) was reported (39,40). Because ubiquitously expressed protein-disulfide isomerases such as ERp57 cooperate together with ER lectin chaperones and help in protein disulfide bond formation (41), it is possible that CALR3 cooperates with PDILT or with other protein-disulfide isomerases during quality control in testes.…”

The redox switch that regulates molecular chaperones

“…Binding of EREcontaining DNA to the oestrogen receptor was recently reported to be enhanced by PDI, independent of its isomerase activity, as evidenced by the ability of the -CGHC-sitedirected mutants to also promote binding (58). Also, PDILT, a PDI homolog, without a redox-active -CGHC-, was shown to display chaperone activity, when found to interact with somatostatin (59). In these examples, the activity of the thioredoxin domains is not required and the chaperone activity is independent of the redox environment.…”