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• W2044595128 abstract "The N-ethylmaleimide-sensitive fusion protein (NSF) is a cytoplasmic protein implicated in the fusion of intracellular transport vesicles with their target membranes. NSF is thought to function in the fusion of essentially all types of vesicles, including endoplasmic reticulum, Golgi, and endocytic vesicles, as well as secretory vesicles undergoing regulated fusion (for review see Rothman, J. E.(1994) Nature 372, 55-63). However, little [Medline] experimental evidence exists to address the possibility that organisms might have multiple NSF proteins serving distinct functions in the same or different cells. We previously cloned a neurally expressed Drosophila homolog, dNSF-1 (Ordway, R. W., Pallanck, L., and Ganetzky, B.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5715-5719), and have subsequently identified mutations in this gene that confer an apparent failure of synaptic transmission at elevated temperature (Pallanck, L., Ordway, R. W., and Ganetzky, B.(1995) Nature, 376, 25; Siddiqi, O., and Benzer, S.(1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3253-3257). Here we report that 1) Drosophila contains a second NSF homolog, termed dNSF-2, that exhibits 84% amino acid identity to dNSF-1, 2) dNSF-1 and dNSF-2 display overlapping but different temporal expression, and 3) multiple transcripts are derived from the dNSF-2 gene. These findings raise the possibility that different NSF gene products serve distinct or overlapping functions within the organism. The N-ethylmaleimide-sensitive fusion protein (NSF) is a cytoplasmic protein implicated in the fusion of intracellular transport vesicles with their target membranes. NSF is thought to function in the fusion of essentially all types of vesicles, including endoplasmic reticulum, Golgi, and endocytic vesicles, as well as secretory vesicles undergoing regulated fusion (for review see Rothman, J. E.(1994) Nature 372, 55-63). However, little [Medline] experimental evidence exists to address the possibility that organisms might have multiple NSF proteins serving distinct functions in the same or different cells. We previously cloned a neurally expressed Drosophila homolog, dNSF-1 (Ordway, R. W., Pallanck, L., and Ganetzky, B.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5715-5719), and have subsequently identified mutations in this gene that confer an apparent failure of synaptic transmission at elevated temperature (Pallanck, L., Ordway, R. W., and Ganetzky, B.(1995) Nature, 376, 25; Siddiqi, O., and Benzer, S.(1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3253-3257). Here we report that 1) Drosophila contains a second NSF homolog, termed dNSF-2, that exhibits 84% amino acid identity to dNSF-1, 2) dNSF-1 and dNSF-2 display overlapping but different temporal expression, and 3) multiple transcripts are derived from the dNSF-2 gene. These findings raise the possibility that different NSF gene products serve distinct or overlapping functions within the organism. INTRODUCTIONProgress in understanding the mechanisms by which intracellular transport vesicles are targeted to and fuse with the appropriate target membrane has led to the characterization of a protein complex thought to perform these functions. This complex includes the cytosolic NSF 1The abbreviations used are: NSFN-ethylmaleimide-sensitive fusion proteinSNAPsoluble NSF attachment proteinSNARESNAP receptorPCRpolymerase chain reactionkbkilobase(s)dNSFDrosophila NSF. and soluble NSF attachment proteins (SNAPs), the SNAP receptors (SNAREs) present on the vesicle and target membranes, and several other proteins (reviewed in (1Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar)). The SNARE hypothesis states that each specific type of vesicle carries a unique set of SNARE protein isoforms (the vesicle SNARE) that is complementary to a unique SNARE on the appropriate target membrane (the target SNARE) and that interactions of these SNAREs are responsible for the specificity of vesicle targeting(1Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). After targeting, the SNAREs are thought to assemble with the cytosolic NSF and SNAP proteins to form a complex that may mediate fusion.Thus the presence of distinct isoforms of the SNARE proteins is essential to the current hypothesis for targeting and fusion mechanisms. In contrast, NSF has been thought to serve a general function(1Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). The results presented here demonstrate the existence of a second NSF gene in Drosophila and raise the possibility that multiple NSF proteins may serve distinct functions.EXPERIMENTAL PROCEDURESCloning and Sequencing of a Novel Drosophila NSFDegenerate oligonucleotide primers corresponding to the conserved NSF peptide sequences HIIIFDE and VIGMTNR were used in PCR to amplify sequences from Drosophila melanogaster genomic DNA. PCR was carried out for 30 cycles of 1 min at 94°C, 2 min at 45°C, and 3 min at 72°C in a Perkin-Elmer thermal cycler. The primer corresponding to HIIIFDE contained a SalI linker sequence, and the primer corresponding to VIGMTNR contained an EcoRI linker. These sites were used to clone the 0.16-kb PCR product into a pBluescript vector (Stratagene) for sequencing. A 32P-labeled probe corresponding to the 0.16-kb sequence was used to screen a D. melanogaster (Oregon R) genomic DNA library (constructed by A. Kamb). The hybridizations were carried out in 6 × SSPE and 50% formamide at 42°C, and the filters were washed in 0.2 × SSC at 60°C. Southern analysis of genomic DNA and of the genomic clones (data not shown) identified a 4.2-kb EcoRI restriction fragment containing the sequence amplified by PCR. This EcoRI fragment was used to screen a cDNA library in λ EXLX+ prepared using RNA isolated from 0-24-h Drosophila embryos (library provided by M. Palazzolo and associates, California Institute of Technology). Screening conditions were as described previously(5Ramaswami M. Tanouye M.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2079-2082Crossref PubMed Scopus (81) Google Scholar). A 1.3-kb SacI fragment containing the 5′ end of the largest embryonic cDNA (G1-2) was then used to screen a cDNA library prepared from adult Drosophila head transcripts (library provided by Tom Schwarz, Stanford University) as described(2Ordway R.W. Pallanck L. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5715-5719Crossref PubMed Scopus (61) Google Scholar). Following purification of phage, cDNAs from the embryonic library were subcloned into a pBluescript vector, and those from the head library were converted to pBluescript plasmid form by autoexcision using the Exassist helper phage system (Stratagene). Sequencing of cDNAs and the 0.16-kb PCR product was carried out either manually (Sequenase kit, U. S. Biochemical Corp.) or using an automated sequencer. The dNSF-2 amino acid sequence (Fig. 1) was primarily derived from the partial open reading frame contained within the embryonic cDNA, G1-2. The amino-terminal 92 amino acids were obtained from an overlapping head library cDNA, dN2.14, which extends the open reading frame in the 5′ direction. Sequences were analyzed using the PILEUP and DISTANCES programs of the Genetics Computer Group (Madison, WI) software package (6Devereux J. Haeberli P. Smithies O. Nucleic Acids Res. 1984; 12: 387-395Crossref PubMed Scopus (11516) Google Scholar).Northern and Chromosomal in Situ Analysis5 μg of poly(A)+ RNA isolated as described (2Ordway R.W. Pallanck L. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5715-5719Crossref PubMed Scopus (61) Google Scholar) from wild type (Canton S) adults, third instar larvae, and mixed stage embryos was separated on a 0.65 M formaldehyde-agarose gel and transferred to nylon membrane (Hybond-N, Amersham Corp.). The blot was hybridized with a 32P-labeled probe derived from either the dNSF-1 cDNA, dN20(2Ordway R.W. Pallanck L. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5715-5719Crossref PubMed Scopus (61) Google Scholar), or the dNSF-2 embryonic cDNA, G1-2, and washed according to the manufacturer's instructions (Hybond-N). In situ hybridization to polytene chromosomes was carried out using the 4.2-kb genomic EcoRI fragment as a probe. The fragment was biotinylated by random primer labeling using biotinylated dUTP in place of dTTP. After binding to streptavidin-conjugated HRP, the hybridization signal was visualized as a peroxidase reaction product using the Detek I HRP kit (ENZO Biochem).RESULTS AND DISCUSSIONDrosophila genomic DNA sequences related to NSF were amplified by PCR using degenerate oligonucleotides corresponding to the conserved NSF amino acid sequences HIIIFDE and VIGMTNR. A 0.16-kb PCR product was cloned, and subsequent sequence analysis showed that it encodes a polypeptide with homology to the previously identified Drosophila NSF (dNSF). The nucleotide sequence of the amplification product was determined to be 86% identical to the corresponding region of dNSF, suggesting that it was derived from a second Drosophila NSF gene. To characterize this putative second gene further, the PCR product was used as a probe to obtain genomic DNA clones and embryonic cDNA clones as described under “Experimental Procedures.” Sequence analysis of two embryonic cDNA clones confirmed that they were derived from a novel Drosophila NSF gene, termed dNSF-2. In situ hybridization to polytene chromosomes localized the dNSF-2 gene to position 87F14-15 on the third chromosome.Since none of the embryonic cDNAs included the 5′ end of the open reading frame, cDNAs extending further 5′ were identified. A probe corresponding to the 5′ most sequence from the largest cDNA, G1-2, was used to perform a high stringency screen of 350,000 recombinant phage from an adult Drosophila head cDNA library. The 43 hybridizing clones detected could be subdivided into two classes: a strongly hybridizing class consisting of 3 members (7% of the total) and a weakly hybridizing class consisting of 40 members (93% of the total). Restriction mapping of two of the strongly hybridizing clones indicated that both were derived from the dNSF-2 gene. In contrast, analysis of seven clones from the weakly hybridizing class indicated that only one was derived from dNSF-2, while the remaining six were from the previously identified dNSF gene, now designated dNSF-1. Thus the cDNAs obtained from the head library using a dNSF-2 probe were predominantly dNSF-1 cDNAs detected by weak cross-hybridization at high stringency. The dNSF-2 gene was not detected as a distinct gene in our previous study(2Ordway R.W. Pallanck L. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5715-5719Crossref PubMed Scopus (61) Google Scholar), apparently due to the cross-hybridization between dNSF-1 and dNSF-2 at high stringency and the predominance of dNSF-1 cDNAs in the head library.Restriction mapping of the largest of the head library dNSF-2 cDNAs, a 3.4-kb clone designated dN2.14, indicated that it extends further 5′ than the largest embryonic cDNA (G1-2). The 5′ end of dN2.14 was sequenced through the region of overlap with G1-2 (see “Experimental Procedures”), and the composite amino acid sequence of dNSF-2 is shown in alignment with dNSF-1 in Fig. 1. On the basis of this alignment the two Drosophila NSFs exhibit 84% amino acid identity. An alignment of dNSF-2 with Chinese hamster ovary NSF and the sec18 gene product (not shown) shows that dNSF-2 is 63 and 42% identical to these polypeptides, respectively. For comparison, the dNSF-1 amino acid sequence is 62 and 42% identical to Chinese hamster ovary NSF and sec18, respectively(2Ordway R.W. Pallanck L. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5715-5719Crossref PubMed Scopus (61) Google Scholar).To compare the temporal expression patterns of the dNSF-1 and dNSF-2 genes, probes derived from each of these genes were used to carry out Northern analysis of mRNA from Drosophila adults, larvae, and embryos (Fig. 2). A single dNSF-1 transcript of approximately 3.2 kb was found to be abundant in adult mRNA but detected at substantially lower levels in larvae and embryos. Expression of dNSF-2 was assessed using the same blot and was found to differ from that of dNSF-1 in two ways. First, multiple transcripts are derived from the dNSF-2 gene, ranging in size from 2.4 to 3.4 kb. Most prominent in this group are bands of 2.4, 2.8, 3.0, and 3.4 kb. Second, dNSF-2 differs from dNSF-1 in that it is expressed at similar levels in adults and larvae, with little or no expression detected in embryos.Figure 2:Northern analysis of poly(A)+ RNA obtained from wild type (Canton S) adults (A), third instar larvae (L), and embryos (E) using probes derived from either dNSF-1 or dNSF-2. The same blot was used in both experiments. Each lane contains 5 μg of poly(A)+ RNA. Size units are kb.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Our previous work has shown that mutations in the dNSF-1 gene are responsible for the comatose temperature-sensitive paralytic phenotype(3Pallanck L. Ordway R.W. Ganetzky B. Nature. 1995; 376: 25Crossref PubMed Scopus (102) Google Scholar), which includes an apparent failure of synaptic transmission at an elevated temperature(4Siddiqi O. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3253-3257Crossref PubMed Scopus (198) Google Scholar). These results, along with functional studies of NSF in other systems, imply a role for dNSF-1 in the regulated exocytosis of synaptic vesicles. The results presented here demonstrate that a second Drosophila NSF, dNSF-2, exhibits 84% amino acid identity with dNSF-1 and that the dNSF-2 gene gives rise to multiple transcripts. Thus at least two Drosophila NSF proteins are derived from these dNSF genes, and future work will assess whether the heterogeneity of dNSF-2 transcripts produces multiple dNSF-2 protein isoforms or serves other functions. We also report here that the temporal expression of dNSF-2 partially overlaps with that of dNSF-1. The abundant expression of both dNSF genes in the adult suggests that both may play important functional roles at this stage. It will be of great interest to determine whether these putative roles are executed in the same or different cell types. It is also of interest to note that the dNSF-1 gene is expressed at much higher levels in adults than in larvae, whereas similar levels of dNSF-2 transcript are detected at these two developmental stages. Furthermore, a low level of dNSF-1 transcript but little or no expression of dNSF-2 is detected in embryos, and the possible existence of additional dNSF genes is now being investigated. The developmental regulation of dNSF gene expression raises the possibility that different NSF functional requirements arise during development and are served by specific NSF gene products. Thus while other studies have suggested that a single NSF protein might serve a universal role in the fusion of intracellular vesicles with target membranes(1Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar), this study suggests more complexity in NSF function. The presence of closely related NSF homologs in Drosophila also raises the possibility that similar complexity exists in vertebrates, a possibility that may explain the apparent existence of multiple secretory mechanisms in some cells (for example, see (7Ikonen E. Tagaya M. Ullrich O. Montecucco C. Simmons K. Cell. 1995; 81: 571-580Abstract Full Text PDF PubMed Scopus (220) Google Scholar)). Further genetic, molecular, and functional analysis will now begin to define the roles of these NSF proteins. INTRODUCTIONProgress in understanding the mechanisms by which intracellular transport vesicles are targeted to and fuse with the appropriate target membrane has led to the characterization of a protein complex thought to perform these functions. This complex includes the cytosolic NSF 1The abbreviations used are: NSFN-ethylmaleimide-sensitive fusion proteinSNAPsoluble NSF attachment proteinSNARESNAP receptorPCRpolymerase chain reactionkbkilobase(s)dNSFDrosophila NSF. and soluble NSF attachment proteins (SNAPs), the SNAP receptors (SNAREs) present on the vesicle and target membranes, and several other proteins (reviewed in (1Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar)). The SNARE hypothesis states that each specific type of vesicle carries a unique set of SNARE protein isoforms (the vesicle SNARE) that is complementary to a unique SNARE on the appropriate target membrane (the target SNARE) and that interactions of these SNAREs are responsible for the specificity of vesicle targeting(1Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). After targeting, the SNAREs are thought to assemble with the cytosolic NSF and SNAP proteins to form a complex that may mediate fusion.Thus the presence of distinct isoforms of the SNARE proteins is essential to the current hypothesis for targeting and fusion mechanisms. In contrast, NSF has been thought to serve a general function(1Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). The results presented here demonstrate the existence of a second NSF gene in Drosophila and raise the possibility that multiple NSF proteins may serve distinct functions." @default.
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• W2044595128 title "Distinct Roles for N-Ethylmaleimide-sensitive Fusion Protein (NSF) Suggested by the Identification of a Second Drosophila NSF Homolog" @default.
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