-
[show abstract]
[hide abstract]
ABSTRACT: The GTPase-activating proteins (GAPs) accelerate the hydrolysis of GTP to GDP by small GTPases. The GTPases play diverse roles in many cellular processes, including proliferation, cell motility, endocytosis, nuclear import/export, and nuclear membrane formation. Little is known about GAP-domain proteins in spermatogenesis. We isolated a novel RhoGAP domain-containing tGAP1 protein from male germ cells that exhibits unusual properties. The tGAP1 is expressed at low levels in early spermatogonia. Robust transcription initiates in midpachytene spermatocytes and continues after meiosis. The 175-kDa tGAP1 protein localizes to the cytoplasm of spermatocytes and to the cytoplasm and nucleus in spermatids. The protein contains four GAP domain-related sequences, in contrast to all other GAP proteins that harbor one such domain. No activity toward RhoA, Rac1, or Cdc42 could be detected. Results of transfection studies in various somatic cells indicated that low-level tGAP1 expression significantly slows down the cell cycle. Expression of higher levels of tGAP1 by infection of somatic cells with recombinant adenoviruses demonstrated that tGAP1 efficiently induces apoptosis, which to our knowledge is the first such demonstration for a RhoGAP protein. Based on its subcellular location in spermatids and its activity, tGAP1 may play a role in nuclear import/export.
Biology of Reproduction 01/2005; 71(6):1980-90. · 4.01 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Outer dense fibers (ODFs) and the fibrous sheath (FS) are unique structures of the mammalian sperm tail. Recently, progress has been made in the molecular cloning of ODF and FS proteins, and because of this, questions addressing the morphogenesis and underlying protein network that make up sperm tail structures and their function can now be addressed. Using the N-terminal leucine zipper motif of the major ODF protein ODF1, we had previously isolated interacting proteins Odf2, Spag4, and Spag5. We report here a yeast two-hybrid strategy to isolate a novel rat testicular protein, OIP1, that binds to the evolutionarily conserved Cys-Gly-Pro repeats in the C-terminus of ODF1. OIP1 is expressed in round spermatids as well as in spermatocytes and several somatic tissues, albeit at a lower level. No expression was detectable in epididymis, heart, and smooth muscle. OIP1 protein localizes to the sperm tail in a pattern expected for an ODF1-interacting protein. OIP1 belongs to the family of RING finger proteins of the H2 subclass. Deletion of the putative RING motif significantly decreased binding to ODF1. Genomic analysis of rat Oip1 and Oip1 homologs indicates that Oip1 is highly conserved. Oip1 is subject to differential splicing and alternative polyadenylation events. It is interesting that Oip1 mRNAs have been reported that lack the exon encoding the putative RING finger.
Biology of Reproduction 03/2003; 68(2):543-52. · 4.01 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In an effort to define the molecular basis for morphogenesis of major sperm tail structures, including outer dense fibers, we recently cloned the Spag5 gene by virtue of its strong and specific leucine-zipper-mediated interaction with Odf1, the 27-kDa major outer dense fiber protein. Spag5 is expressed during meiosis and in round spermatids and is similar, if not identical, to Deepest, a putative spindle pole protein. Here we report the disruption of the Spag5 gene by homologous recombination. Spag5-null mice lack Spag5 mRNA and protein. However, male mice are viable and fertile. Analysis of the process of spermatogenesis and sperm produced in Spag5-null mice did not reveal a major phenotype as a consequence of the knockout event. This result suggests that if Spag5 plays a role in spermatogenesis it is likely compensated for by unknown proteins.
Molecular and Cellular Biology 05/2002; 22(7):1993-7. · 5.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The study of mammalian sperm tail outer dense fibers (ODF), a structure of unknown function, is hampered by the insoluble
nature of ODF proteins and the availability of only one cloned component, Odf27. We report here the first use of the Odf27
leucine zipper as bait in a yeast two-hybrid screen to isolate a novel testis-specific protein whose interaction with Odf27
depends critically on the Odf27 leucine zipper. We find that the novel gene, 111-450, encodes a product that localizes to
ODF as determined by fluorescence microscopy and immunoelectron microscopy and that the gene 111-450 product is identical
to the major ODF protein, Odf84. Interestingly, Odf84 contains two C-terminal leucine zippers, and we demonstrate that all
leucine residues in the upstream leucine zipper are required for interaction with Odf27, demonstrating the strategic validity
of our approach. The use of the yeast screening approach to isolate leucine zipper containing proteins should be useful in
other systems, and our findings have implications for ODF structural models.
Journal of Biological Chemistry 03/1997; 272(10):6105-6113. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The c-mos gene is transcribed in male and female germ cells, in differentiating myoblasts and in 3T3 cells from cell-specific promoters. We characterized the rat testis promoter, which contains a TATA-box and one binding site for a testis-specific transcription factor TTF-D, as well as a region which can act as enhancer, which is located approx. 2 kb upstream of the c-mos AUG start codon. It binds three factors at sites I, II and III as determined in DNAse I footprint assays. We demonstrated that a member of the NF-1/CTF family of transcription factors binds site II. Here we report the cloning of the protein that binds to enhancer site III. This protein is the rat homolog of human hCut/CDP, mouse Cux/CDP and canine Clox. hCut/Cux/CDP/Clox (hereafter called Cux/CDP), a 160 kDa protein containing multiple repeats and a homeodomain, negatively regulates the mammalian c-myc, gp91-phox and N-Cam genes. Using bacterially produced murine GST-Cux fusion proteins and GST-Cux deletion mutants, we find that Cux repeat CR3 and the homeodomain are both required for efficient binding to enhancer site III. Mouse lung and testis nuclear Cux/CDP bind to site III as determined in electrophoretic gel mobility supershift assays using two different anti-hCut specific monoclonal antibodies. Transfections of CAT constructs containing the enhancer fragment linked to a minimal promoter demonstrated that Cux/CDP represses c-mos enhancer activity.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression.
