A Drosophila homolog of the membrane fusion protein CDC48/p97 has been cloned. The open reading frame of the Drosophila homolog encodes an 801 amino acid long protein (TER94), which shows high similarity to the known CDC48/p97 sequences. The chromosomal position of TER94 is 46 C/D. TER94 is expressed in embryo, in pupae and in the adult, but is suppressed in larva. In the adults, the immunoreactivity is exclusively present in the head and in the gonads of both sexes. In the head the most striking staining is observed in the entire neuropil of the mushroom body and in the antennal glomeruli. Besides TER94, sex-specific forms are also detected in adult gonads: p47 in the ovaries and p98 in the testis. TER94/p47 staining is observed in the nurse cells and often in the oocytes, while TER94/p98 staining is present in the sperm bundles. On the basis of the TER94 distribution it is suggested that TER94 functions in the protein transport utilizing endoplasmic reticulum and Golgi derived vesicles (Pinter, 1998).
The Drosophila fusome is a germ cell-specific organelle assembled from membrane skeletal proteins and membranous vesicles. Mutational studies that have examined inactivating alleles of fusome proteins indicate that the organelle plays central roles in germ cell differentiation. Although mutations in genes encoding skeletal fusome components prevent proper cyst formation, mutations in the bag-of-marbles gene disrupt the assembly of membranous cisternae within the fusome and block cystoblast differentiation altogether. To understand the relationship between fusome cisternae and cystoblast differentiation, attempts have been made to identify other proteins in this network of fusome tubules. Evidence is presented that the fly homolog of the transitional endoplasmic reticulum ATPase (TER94) is one such protein. The presence of TER94 suggests that the fusome cisternae grow by vesicle fusion and are a germ cell modification of endoplasmic reticulum. Fusome association of TER94 is Bam-dependent, suggesting that cystoblast differentiation may be linked to fusome reticulum biogenesis (Leon, 1999).
Antisera raised against a TER94 internal peptide reacts with bands of 94,000 Da in wild-type ovarian extracts and 57,000 Da in Escherichia coli cells expressing a fragment of TER94 as a GST-fusion protein. Both Cdc48p and vertebrate TERs oligomerize to form homohexameric complexes. When ovarian extracts were analyzed on native sucrose gradients, the peak of TER94 from flies sedimented was Mr ~500,000, which is close to the expected size (Mr ~530,000) for a homohexameric complex (Leon, 1999).
TER94 protein is present in both ovarian germ cells and somatic cells. TER94 is largely cytoplasmic in follicle and germ cells. Significantly, germ cells often contain one or several especially intense fluorescent signals, suggesting that TER94 is distributed unevenly in the cytoplasm. In cystocytes in germarial Region 1, these are usually somewhat diffuse bright regions, whereas in more mature cystocytes the bright spots are more sharply defined (Leon, 1999).
The number and positions of the TER-enriched regions suggest that they might correspond to fusomes. Stem cell fusomes in germ cells nearest the anterior tip appear as a single dot of intense staining, whereas those in a more posterior position (i.e. more mature cysts) contain elongated, branched fusomes. Precise colocalization of TER94 and Hu-li tao shao is strongest in Region 1 germ cells and declines in regions containing mature cysts. Because a fraction of TER is nuclear in yeast and mammals, Drosophila nuclei were examined closely. Most germ cell nuclei are faintly TER94 positive. Many examples of strong nuclear and perinuclear staining have been found in nonovarian somatic cells in larvae and adults (Leon, 1999).
Fusomes are the primary site of ER-like cisternae in young germ cells. If TER94 enrichment in fusomes represents accumulation at the fusome reticulum, TER94 distribution might be altered when the reticulum is not properly assembled. Bam is a fusome-associated protein and bam mutant fusomes are deficient in cisternae. The distribution of TER94 protein was examined in bam germ cells; it is distributed uniformly without signs of enrichment at the site of fusomes as is observed in wild-type germaria. Indeed, when the bam stem cell fusomes are visualized with Hts antibodies, it is clear that TER94 is no more abundant within or near stem cell fusomes than in any other cytoplasmic regions. Consistent with this conclusion, the merged images of TER94 and Hts distributions do not show immunofluorescent overlap, indicating that bam fusomes do not accumulate detectable TER94 (Leon, 1999).
TER94 is also enriched at a few sites that do not correspond to fusomes. It is speculated that these may be sites of Golgi bodies or transport vesicles, although unambiguous identification requires additional reagents as markers. These extrafusome sites of TER94 enrichment are also abolished in bam mutant cells (Leon, 1999).
The observation that TER94 fusome association is linked to Bam function can be explained by either a direct or indirect Bam dependent mechanism. Although loss of bam function might block fusome reticulum assembly before TER94 arrival, it is also possible that Bam recruits TER94 to the reticulum as part of the assembly process. This hypothesis has been difficult to test because Bam is a low-abundance protein in ovaries, and in vitro assays for Bam and TER94 interaction have produced inconsistent results. The interaction of Bam and TER94 as two-hybrid partners supports the hypothesis of in vivo interaction. Finding the Drosophila homolog of the S. cerevisiae protein Ufd3p as a second Bam interacting protein strengthens the significance of the Bam-TER94 interaction. Ufd3p and the yeast TER (i.e., Cdc48p) interact with one another directly. Ufd3p is required for efficient organelle vesicle fusion (Leon, 1999 and references therein).
A genetic screen was carried out in Drosophila to identify mutations that disrupt the localization of Oskar mRNA during oogenesis. Based on the hypothesis that some cytoskeletal components that are required during the mitotic divisions will also be required for Oskar mRNA localization during oogenesis, a screen was carried out for P-element insertions in genes that slow down the blastoderm mitotic divisions. A secondary genetic screen was used to generate female germ-line clones of these potential cell division cycle genes and to identify those that cause the mislocalization of Oskar mRNA. Mutations were identified in ter94 that disrupt the localization of Oskar mRNA to the posterior pole of the oocyte. Ter94 is a member of the CDC48p/VCP subfamily of AAA proteins which are involved in homotypic fusion of the endoplasmic reticulum during mitosis. Consistent with the function of the yeast ortholog, ter94-mutant egg chambers are defective in the assembly of the endoplasmic reticulum. A test was carried out to see whether other membrane biosynthesis genes are required for localizing Oskar mRNA during oogenesis. Ovaries that are mutant for syntaxin-1a, rop, and synaptotagmin are also defective in Oskar mRNA localization during oogenesis (Ruden, 2000).
In order to identify new genes required for OSK mRNA localization, OSK localization defects in egg chambers were sought in mutants for cell division cycle (CDC) genes that had been isolated in a 'mitotic delay-dependent survival' (MDDS) genetic screen. The rationale for this is that many cytoskeletal proteins required for mitotic divisions may also be required for mRNA localization. The advantage of studying the function of CDC genes during oogenesis, in which all of the mitotic divisions occur in region 1 of the germarium, is that later in oogenesis one can analyze the biological functions of the CDC genes independent of their mitotic functions. For example, Klp38B, a chromatin-binding kinesin-like-protein isolated in the MDDS genetic screen, is required not only for chromosome segregation during the meiotic and mitotic divisions, but also for the proper development of the oocyte, possibly by localizing mRNA or protein in the oocyte (Ruden, 2000 and references therein).
Based on the phenotypes of syx-1a, ter94, rop and syt mutant egg chambers, a three-step genetic pathway is proposed for the role of membrane fusion proteins on OSK mRNA localization during oogenesis. (1) Syx-1a is required in stage 1 egg chambers to get OSK mRNA to the oocyte. Syx was originally identified as a Drosophila homolog of a human tSNARE that is required for synaptic vesicle fusion in neurons. Interestingly, Syx5 in humans has recently been shown to be required for TERA-mediated (the human Ter94 ortholog) assembly of Golgi cisternae from mitotic Golgi fragments in vitro (Rabouille, 1998). (2) Ter94 is required to localize OSK mRNA within the oocyte. It is speculated that OSK mRNA might be transported in membranous particles because both the endoplasmic reticulum and OSK mRNA form particulate complexes in ter94-mutant egg chambers. (3) The final step in OSK mRNA localization is anchoring the mRNA to the posterior pole of the oocyte. It is proposed that Rop and Syt are required for this process because rop and syt mutant egg chambers have poorly formed cytoplasmic membranous structure in the oocytes, and, possibly as a result, OSK mRNA fails to remain localized at the posterior pole. Rop is a Drosophila homolog of yeast Sec1 and vertebrate n-Sec1/Munc-18 proteins and is a negative regulator of neurotransmitter release in vivo. Syt controls and modulates synaptic vesicle fusion in a Ca2+ dependent manner. It is concluded that many synaptic vesicle fusion proteins also function during other cellular processes such as OSK mRNA localization during oogenesis (Ruden, 2000 and references therein).
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