The Drosophila Cog5 Homologue Is Required for Cytokinesis, Cell Elongation, and Assembly of Specialized Golgi Architecture during Spermatogenesis

The multisubunit conserved oligomeric Golgi (COG) complex has been shown previously to be involved in Golgi function in yeast and mammalian tissue culture cells. Despite this broad conservation, several subunits, including Cog5, were not essential for growth and showed only mild effects on secretion when mutated in yeast, raising questions about what functions these COG complex subunits play in the life of the cell. Here, we show that function of the gene four way stop (fws), which encodes the Drosophila Cog5 homologue, is necessary for dramatic changes in cellular and subcellular morphology during spermatogenesis. Loss-of-function mutations in fws caused failure of cleavage furrow ingression in dividing spermatocytes and failure of cell elongation in differentiating spermatids and disrupted the formation and/or stability of the Golgi-based spermatid acroblast. Consistent with the lack of a growth defect in yeast lacking Cog5, animals lacking fws function were viable, although males were sterile. Fws protein localized to Golgi structures throughout spermatogenesis. We propose that Fws may directly or indirectly facilitate efficient vesicle traffic through the Golgi to support rapid and extensive increases in cell surface area during spermatocyte cytokinesis and polarized elongation of differentiating spermatids. Our study suggests that Drosophila spermatogenesis can be an effective sensitized genetic system to uncover in vivo functions for proteins involved in Golgi architecture and/or vesicle transport

Identification of Sec36p, Sec37p, and Sec38p: Components of Yeast Complex That Contains Sec34p and Sec35p

The Saccharomyces cerevisiae proteins Sec34p and Sec35p are components of a large cytosolic complex involved in protein transport through the secretory pathway. Characterization of a new secretion mutant led us to identify SEC36, which encodes a new component of this complex. Sec36p binds to Sec34p and Sec35p, and mutation of SEC36 disrupts the complex, as determined by gel filtration. Missense mutations of SEC36 are lethal with mutations in COPI subunits, indicating a functional connection between the Sec34p/sec35p complex and the COPI vesicle coat. Affinity purification of proteins that bind to Sec35p-myc allowed identification of two additional proteins in the complex. We call these two conserved proteins Sec37p and Sec38p. Disruption of either SEC37 or SEC38 affects the size of the complex that contains Sec34p and Sec35p. We also examined COD4, COD5, and DOR1, three genes recently reported to encode proteins that bind to Sec35p. Each of the eight genes that encode components of the Sec34p/sec35p complex was tested for its contribution to cell growth, protein transport, and the integrity of the complex. These tests indicate two general types of subunits: Sec34p, Sec35p, Sec36p, and Sec38p seem to form the essential core of a complex to which Sec37p, Cod4p, Cod5p, and Dor1p seem to be peripherally attached

Genomic Screen for Vacuolar Protein Sorting Genes in Saccharomyces cerevisiae

The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature α-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30°C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process

CASP, the Alternatively Spliced Product of the Gene Encoding the CCAAT-Displacement Protein Transcription Factor, Is a Golgi Membrane Protein Related to Giantin

Large coiled-coil proteins are being found in increasing numbers on the membranes of the Golgi apparatus and have been proposed to function in tethering of transport vesicles and in the organization of the Golgi stack. Members of one class of Golgi coiled-coil protein, comprising giantin and golgin-84, are anchored to the bilayer by a single C-terminal transmembrane domain (TMD). In this article, we report the characterization of another mammalian coiled-coil protein, CASP, that was originally identified as an alternatively spliced product of the CUTL1 gene that encodes CCAAT-displacement protein (CDP), the human homologue of the Drosophila homeodomain protein Cut. We find that the Caenorhabditis elegans homologues of CDP and CASP are also generated from a single gene. CASP lacks the DNA binding motifs of CDP and was previously reported to be a nuclear protein. Herein, we show that it is in fact a Golgi protein with a C-terminal TMD and shares with giantin and golgin-84 a conserved histidine in its TMD. However, unlike these proteins, CASP has a homologue in Saccharomyces cerevisiae, which we call COY1. Deletion of COY1 does not affect viability, but strikingly restores normal growth to cells lacking the Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptor Gos1p. The conserved histidine is necessary for Coy1p's activity in cells lacking Gos1p, suggesting that the TMD of these transmembrane Golgi coiled-coil proteins is directly involved in their function