The vesicular trafficking inside eukaryotic cells is a highly regulated process that requires specificity at each transport step. The selective interaction between donor and acceptor membranes is mediated by tethering factors, among which GARP (Golgi associated retrograde protein) complex. The vacuolar protein sorting Vps54 is a subunit of GARP and in yeast is involved in the retrograde transport of early endosomes to the trans Golgi network. In the mouse, a single point mutation near the C-terminus of Vps54 (L967Q) gives rise to the Wobbler phenotype, characterized by defects in motor neuron function and male sterility. We took advantage of this mutant variant to investigate more in detail the role of Vps54 in vesicular trafficking of mammalian cells. In cultured HEK293 cells, Vps54-GFP colocalizes with Golgi and partially with endosomal compartment. The localization of the wobbler variant is comparable with that of wt protein, even if Vps54L967Q-GFP is slightly more diffuse into the cytoplasm. This result supports the literature data suggesting that wobbler Vps54 retains activity in trafficking. Since wobbler disease specifically affects spermatogenesis, we focused our attention on Vps54 in developing mouse germ cells. In the physiological contest, we demonstrated that Vps54 is involved in acrosome biogenesis. The acrosome is an acidic vesicle of not better-identified origin, indispensable for fertilization. Wobbler spermatozoa are resulted to lack a true acrosome: during sperm cell differentiation Vps54(L967Q) marks numerous punctuated vesicles that, on the contrary of the wt Vps54-labeled ones, are not able to coalesce and develop into the acrosome. These results were confirmed by double immunostaining with USP8, a deubiquitinating enzyme marker of the endocytic pathway. We investigated by exploiting recombinant proteins if the absence of an acrosome could depend on the failure of Vps54L967Q to interact with sperm factors involved in trafficking machinery
Paiardi, C., Ceriani, M., Martegani, E., Berruti, G. (2011). Failure of acrosomogenesis in germ cells of the wobbler mutant reflects impaired vesicle trafficking. In FEBS journal, vol.278, issue supplement s1,Article first published online: 1 JUL 2011 (pp.415-415). WILEY-BLACKWELL [10.1111/j.1742-4658.2011.08137.x].
Failure of acrosomogenesis in germ cells of the wobbler mutant reflects impaired vesicle trafficking
CERIANI, MICHELA;MARTEGANI, ENZO;
2011
Abstract
The vesicular trafficking inside eukaryotic cells is a highly regulated process that requires specificity at each transport step. The selective interaction between donor and acceptor membranes is mediated by tethering factors, among which GARP (Golgi associated retrograde protein) complex. The vacuolar protein sorting Vps54 is a subunit of GARP and in yeast is involved in the retrograde transport of early endosomes to the trans Golgi network. In the mouse, a single point mutation near the C-terminus of Vps54 (L967Q) gives rise to the Wobbler phenotype, characterized by defects in motor neuron function and male sterility. We took advantage of this mutant variant to investigate more in detail the role of Vps54 in vesicular trafficking of mammalian cells. In cultured HEK293 cells, Vps54-GFP colocalizes with Golgi and partially with endosomal compartment. The localization of the wobbler variant is comparable with that of wt protein, even if Vps54L967Q-GFP is slightly more diffuse into the cytoplasm. This result supports the literature data suggesting that wobbler Vps54 retains activity in trafficking. Since wobbler disease specifically affects spermatogenesis, we focused our attention on Vps54 in developing mouse germ cells. In the physiological contest, we demonstrated that Vps54 is involved in acrosome biogenesis. The acrosome is an acidic vesicle of not better-identified origin, indispensable for fertilization. Wobbler spermatozoa are resulted to lack a true acrosome: during sperm cell differentiation Vps54(L967Q) marks numerous punctuated vesicles that, on the contrary of the wt Vps54-labeled ones, are not able to coalesce and develop into the acrosome. These results were confirmed by double immunostaining with USP8, a deubiquitinating enzyme marker of the endocytic pathway. We investigated by exploiting recombinant proteins if the absence of an acrosome could depend on the failure of Vps54L967Q to interact with sperm factors involved in trafficking machinery
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