Developmental Genetics of Secretory Vesicle Acidification DuringCaenorhabditis elegansSpermatogenesis

Abstract

Secretory vesicles are used during spermatogenesis to deliver proteins to the cell surface. In Caenorhabditis elegans, secretory membranous organelles (MO) fuse with the plasma membrane to transform spermatids into fertilization-competent spermatozoa. We show that, like the acrosomal vesicle of mammalian sperm, MOs undergo acidification during development. Treatment of spermatids with the V-ATPase inhibitor bafilomycin blocks both MO acidification and formation of functional spermatozoa. There are several spermatogenesis-defective mutants that cause defects in MO morphogenesis, including spe-5. We determined that spe-5, which is on chromosome I, encodes one of two V-ATPase B paralogous subunits. The spe-5 null mutant is viable but sterile because it forms arrested, multi-nucleate spermatocytes. Immunofluorescence with a SPE-5-specific monoclonal antibody shows that SPE-5 expression begins in spermatocytes and is found in all subsequent stages of spermatogenesis. Most SPE-5 is discarded into the residual body during spermatid budding, but a small amount remains in budded spermatids where it localizes to MOs as a discrete dot. The other V-ATPase B subunit is encoded by vha-12, which is located on the X chromosome. Usually, spe-5 mutants are self-sterile in a wild-type vha-12 background. However, an extrachromosomal transgene containing wild-type vha-12 driven by its own promoter allows spe-5 mutant hermaphrodites to produce progeny, indicating that VHA-12 can at least partially substitute for SPE-5. Others have shown that the X chromosome is transcriptionally silent in the male germline, so expression of the autosomally located spe-5 gene ensures that a V-ATPase B subunit is present during spermatogenesis. V ESICULAR organelles in eukaryotic cells frequently maintain an acidic pH (reviewed by Paroutis et al. 2004) that is created by the vacuolar H+-ATPase (V-ATPase). The V-ATPase is a large (910-kDa) molecular machine that couples ATP hydrolysis to the movement of protons across biological membranes. The V-ATPase has a V 0 -sector that creates the pore-for-proton translocation through the lipid bilayer and a V 1 -sector, located in the cytoplasm, that is the site of ATP hydrolysis. Each V-ATPase holoenzyme is composed of 14 different subunits, some of which are present in multiple copies (reviewed by Toei et al. 2010). In yeast, there is one gene for each V-ATPase subunit, except for the "a" subunit, which is encoded by two genes (reviewed by Kane 2006). The physiological properties of the V-ATPase are in part determined by which of these two "a" subunits it contains (Kawasaki-Nishi et al. 2001). In humans and other animals, the "a" and other V-ATPase subunits are encoded by more than one gene (reviewed by Toei et al. 2010). Subunit diversity presumably allows the V-ATPase to be either customized for a specific function or utilized in a tissuespecific fashion.In this article, we use pH-sensitive vital dyes and specific inhibitors to show that sperm-specific MOs use the V-ATPase to acidify their interior a...

V-ATPase V1 Sector Is Required for Corpse Clearance and Neurotransmission in Caenorhabditis elegans

“…The C. elegans genome contains a second locus, called spe-5 (Y110A7A.12), that encodes a V-ATPase B subunit. Because spe-5 mutants are defective for spermatogenesis (see accompanying article by Gleason et al 2012) and vha-12 null mutants are lethal, these loci are not fully redundant and independent functions can be characterized.…”

“…Although C. elegans contains two B subunit paralogs, our functional and expression pattern analyses suggest that VHA-12 is required in essentially every somatic C. elegans cell. By contrast, SPE-5 seems to be largely limited to a requirement in sperm (Gleason et al 2012). It is possible that SPE-5 confers specialized attributes or regulation to the V 1 sector in sperm.…”

“…It is possible that SPE-5 confers specialized attributes or regulation to the V 1 sector in sperm. However, VHA-12 can at least partially compensate for loss of SPE-5 (Gleason et al 2012). …”

“…We also show a previously unrecognized role of the catalytic B subunit in the clearance of apoptotic cell corpses in embryos. vha-12 is thus required broadly, whereas spe-5 is required only during spermiogenesis (Gleason et al 2012). Together these articles suggest that B-subunit diversity in C. elegans may have arisen to escape X chromosome inactivation rather than to provide functional diversity for the V-ATPase proton pump.…”

“…To investigate whether these B-subunit genes have specialized functions, we and the authors of the accompanying article in this issue (Gleason et al 2012) have cloned and characterized mutations in each of these loci. We isolated a null allele and a missense allele in the vha-12 locus, which is on the X chromosome.…”