Submitted August 15, 2007
Returned for revision August 30, 2007
Accepted March 27, 2008
Gamete Biology
RAB2A: A Major Subacrosomal Protein of Bovine Spermatozoa Implicated in Acrosomal Biogenesis
Jeremi R. Mountjoy ,
Wei Xu ,
Dave McLeod ,
David Hyndman ,
and
Richard Oko *
* To whom correspondence should be addressed. E-mail: ro3{at}post.queensu.ca.
Abstract
The perinuclear theca (PT) of mammalian sperm is a unique subcellular structure encapsulating the nucleus. Compositionally the PT is made up of at least six prominent polypeptides (60-, 36-, 31-, 28-, 24-, 15-kDa) of which only two have been sequence identified, as well as many less prominent ones. As an ongoing process in unveiling the protein composition of the PT, we have uncovered the sequence identity of the prominent 24 kDa polypeptide (PT24). Initial N-terminal sequence analysis obtained by Edman degradation suggested PT24 being a RAB2 protein. This was corroborated by MALDI-TOF and Q-TOF mass spectrometric analysis of trypsin-digested fragments of PT24, identifying RAB2A of the RAB2 subfamily as the best sequence match. Q-TOF analysis identified 72% sequence coverage between PT25 and bull, human, mouse or rabbit RAB2A. Since a genome search only identified two RAB2 subfamily members, RAB2A and RAB2B, the 72% sequence coverage of PT24 provides assurance that this protein is RAB2A and not a new RAB2 subfamily member. Furthermore commercial RAB2A antibodies, raised against oligopeptide fragments in the unique C-terminal region of RAB2A, specifically labelled PT24 on westerns of PT extracts. These anti-RAB2A antibodies, along with immune serum we raised and affinity purified against isolated PT24, demonstrated at both light and electron microscope levels that RAB2 is associated with the periphery of the growing proacrosomic and acrosomic vesicles in the Golgi and cap phases of spermiogenesis and consequently assembled as part of the PT. This pattern of subacrosomal assembly is reminiscent of the pathway used by SubH2Bv (PT15), another prominent and exclusive subacrosomal protein, indicating a common route for subacrosomal-PT assembly. Traditionally somatic RAB2 proteins are involved in vesicular transport between the ER and the cis-side of the Golgi apparatus. Our study suggests an unprecedented direction of RAB2A mediated vesicular transport in spermatids during acrosomal biogenesis, from the trans-side of the Golgi apparatus to the nuclear envelope.
Key words:
Gamete Biology •
Testis •
Sperm •
Spermatid •
Spermatogenesis
