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This Article Full Text Full Text (PDF) [Supplemental Figures] All Versions of this Article: 79/2/223    most recent biolreprod.107.065060v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Mountjoy, J. R. Articles by Oko, R. PubMed PubMed Citation Articles by Mountjoy, J. R. Articles by Oko, R. Agricola Articles by Mountjoy, J. R. Articles by Oko, R.

RAB2A: A Major Subacrosomal Protein of Bovine Spermatozoa Implicated in Acrosomal Biogenesis¹

Departments of Anatomy and Cell Biology³ and Biochemistry,⁴ Queen's University, Kingston, Ontario, Canada K7L3N6

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, and 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 that PT24 is a RAB2 protein. This was corroborated by mass spectrometric analyses of trypsin-digested fragments of PT24, identifying RAB2A of the RAB2 subfamily as the best sequence match. Quadrapole/time-of-flight analysis identified 72%% sequence coverage between PT24 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 labeled PT24 on Western blot analysis of PT extracts. These anti-RAB2A antibodies, along with immune serum that 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 endoplasmic reticulum 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.

acrosomal membrane, acrosome formation, gamete biology, perinuclear theca, PT24, RAB2A, sperm, spermatid, spermatogenesis, subacrosomal layer, testis, vesicular transport

¹Supported by a grant (MOP-84440) from the Canadian Institute of Health Research (R.O.). The Queen's Protein Function Discovery Facility was essential for this project and receives infrastructure funding through the Canadian Foundation for Innovation and Ontario Innovation Trust and operating and maintenance support from a Canadian Institutes of Health Research Resource Grant (PRG-80162) and the Faculty of Health Sciences at Queen's University.

Correspondence: ²Richard Oko, Queen's University, Department of Anatomy and Cell Biology, 9th Floor, Botterell Hall, Kingston, Ontario, Canada K7L3N6. FAX: 613 533 2566; e-mail: ro3{at}post.queensu.ca