Characterization of Karyopherin Alpha's Relationship with SubH2Bv as Acrosome-Associated Proteins in Spermiogenesis
Tran, MONG HOA
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Specialized in form and function, the sperm cell is a unique microsystem unto itself where in the cytoskeletal processes and structures of the somatic cell often find new purpose and characteristics within the sperm. Unlike other cells in the human body, this unique cell polarizes and transforms itself from a line of germ cells to evolve into a functional, hydrodynamic haploid spermatozoon. The success of fertilization is dependent on this haploid cell and its specially designed vesicular structure, the acrosome, which provides the leading edge of oocyte penetration. To date, there is little insight into the mechanics of how acrosomic vesicles are successfully targeted and transported to the nuclear envelope and tether to its surface. Our laboratory has identified a novel 15 kDa sperm specific histone variant, SubH2Bv, which possesses a distinct and functional nuclear localization signal (NLS) that associates with the acrosomic vesicle. This study provides evidence that SubH2Bv’s bipartite NLS (an NLS with two basic domains linked together by 10-12 amino acid residues) is responsible for directing acrosomic vesicles to the nuclear envelope using the somatic import receptor, karyopherin alpha (Kap α). Based on bipartite NLS-receptor conventions, where karyopherin alpha is known to specifically associate with this NLS-type, SubH2Bv would be the karyophilic cargo and karyopherin alpha would act as part of the underlying transport mechanism. Western blot analysis and immunohistochemistry characterized Kap α as a membrane-associated sperm protein that is co-localized with SubH2Bv around the proacrosomic granules and the acrosomic vesicle during spermiogenesis. Their co-expression and co-localization, as demonstrated by immunolabelling, suggested a potential binding relationship that was confirmed by a His-tag-recombinant SubH2Bv-pull-down assay. The co-developmental acrosomic expression of Kap α and SubH2Bv in haploid cells, combined with the pull-down evidence of their binding affinity, provides a compelling argument that these two proteins work in concert to traffic the acrosomic vesicles to the nucleus. The exclusion of these two otherwise nuclear proteins from the nucleus, and their co-localization to the subacrosomal region in elongating spermatids, also implies a contingent role for SubH2Bv and Kap α in acrosomal docking, that may involve the classical bipartite/Kap α nuclear import pathway.