(Received for publication, May 18, 1995; and in revised form, June 16, 1995)
From the
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.
Progress 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 ()and soluble NSF attachment proteins (SNAPs), the SNAP
receptors (SNAREs) present on the vesicle and target membranes, and
several other proteins (reviewed in (1) ). 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(1) . 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(1) . 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.
Figure 1: Alignment of the deduced amino acid sequence of dNSF-2 with that of dNSF-1. Amino acid identities are highlighted.
Drosophila 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(2) , 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(2) .
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.
Our previous work has shown that mutations in the dNSF-1 gene are responsible for the comatose temperature-sensitive paralytic phenotype(3) , which includes an apparent failure of synaptic transmission at an elevated temperature(4) . 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(1) , 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 (7) ). Further genetic, molecular, and functional analysis will now begin to define the roles of these NSF proteins.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U09373[GenBank].