Caenorhabditis elegans p97 controls germline-specific sex determination by controlling the TRA-1 level in a CUL-2-dependent manner

Abstract

p97 (CDC-48 inCaenorhabditis elegans) is a ubiquitin-selective AAA (ATPases associated with diverse cellular activities) chaperone and its key function is to disassemble protein complexes. p97 functions in diverse cellular processes including endoplasmic reticulum (ER)-associated degradation, membrane fusion, and meiotic and mitotic progression. However, its cellular functions in development have not yet been clarified. Here, we present data that p97 is involved in the switch from spermatogenesis to oogenesis in the germline of the C. elegans hermaphrodite. We found that the cdc-48.1 deletion mutant produced less sperm than the wild type and thus showed a decreased brood size. The cdc-48.1 mutation suppressed the sperm-overproducing phenotypes of fbf-1 and fem-3(gf) mutants. In addition, the p97/CDC-48-UFD-1-NPL-4 complex interacted with the E3 ubiquitin ligase CUL-2 complex via NPL-4 binding to Elongin C. Furthermore, TRA-1A, which is the terminal effector of the sex determination pathway and is regulated by CUL-2-mediated proteolysis, accumulated in the cdc-48.1 mutant. Proteasome activity was also required for the brood size determination and sperm-oocyte switch. Our results demonstrate that the C. elegans p97/CDC-48-UFD-1-NPL-4 complex controls the sperm-oocyte switch by regulating CUL-2-mediated TRA-1A proteasome degradation.

Characterization of C-terminal adaptors, UFD-2 and UFD-3, of CDC-48 on the polyglutamine aggregation in C. elegans

“…The deletion mutants of cdc-48.1(tm544), ufd-2(tm1380) and ufd-3(tm2915) were obtained from Dr. Shohei Mitani (Tokyo Women's Medical University). XA7200 unc-119(ed3) qaIs7200[unc-119(þ)] and XA7203 unc-119(ed3); cdc-48.1(tm544) qaIs7201[Pcdc-48.1-FLAG::CDC-48.1, unc-119(þ)] were reported previously [31]. To construct transgenic worms expressing expanded polyQ stretches, DNA fragment encoding 40 polyQ stretches fused to GFP (polyQ40::GFP) was cloned and microinjected as described previously [8].…”

“…S2). Previously we have prepared the C. elegans strain XA7203, which expresses FLAG-tagged CDC-48.1 [31]. Lysates were immunoprecipitated with the anti-FLAG(M2) antibody and immunoprecipitates were analyzed by Western blotting by using anti-CDC-48, anti-UFD-2, and anti-UFD-3 antibodies.…”

CDC-48/p97 is required for proper meiotic chromosome segregation via controlling AIR-2/Aurora B kinase localization in Caenorhabditis elegans

“…Cytological observations were carried out as described previously (Sasagawa et al, 2007(Sasagawa et al, , 2009(Sasagawa et al, , 2010). An Olympus Power BX51 microscope equipped with a CoolSnapHQ CCD camera (Roper Scientific) was used.…”

fidgetin homolog FIGL-1, a nuclear-localized AAA ATPase, binds to SUMO

“…Gonads were dissected on poly-L-lysine-coated slides, fixed and immunostained (Sasagawa et al, 2009). Samples were observed under an Olympus Power BX51 microscope.…”

“…Plasmids were verified by DNA sequencing. The constructs for cdc-48.1 and cdc-48.2 were reported previously (Sasagawa et al, 2009). Site-directed mutagenesis of figl-1 and smo-1 was carried out using a QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene).…”