The Sec1/Munc-18 family is an essential factor in both constitutive and regulated secretion, but the physiological role is still unclear. In yeast, 4 Sec1/Munc-18 proteins (Sec1p, Sly1p, Slp1p and Vps45p) are known to function in specific membrane trafficking steps. We have found that C elegans
unc-18 , homologous to Sec1p, is expressed in nervous system and loss of function mutations in this gene result in defects of neurotransmitter release. There are at least 5 members of this family in addition to
unc-18 on the C elegans genome. In this work, we determined the full length cDNA sequence of the C elegans
vps45 C44C1.4 whose orthologous protein in yeast plays an important role in membrane trafficking from Golgi's apparatus to vacuole. A comparison of genomic and cDNA sequences of Ce-
vps45 revealed that 11 exons encoded 547 amino acids having 40 % identity to Vps45, as predicted by Genefinder. The Ce-
vps45::EGFP fusion protein was expressed in all tissues, predominantly in intestinal cells. To get insight into the function of Ce-
vps45 , we isolated a putative null mutant,
tm246 , which had a 1.2 kb deletion covering the first to fourth exons of Ce-
vps45 gene, using TMP/UV mutagenesis. We found that the
tm246 mutation showed a maternal effect ts lethal phenotype, exhibiting developmental arrest in the early larva at 25¼C, while reaching to adulthood and were fertile at 15C. A genomic fragment og 7.5 kb containing the entire ORF complemented
tm246 mutation, resulting in normal growth of transgenic animals at 25C.
tm246 mutation causes amorphous autofluorescense in the intestinal cells instead of intestinal lipofuscin granules, which are thought to be located in secondary lysosomes. When Ce-
vps45 cDNA was specifically expressed in the intestine of
tm246 animals under the
ges-1 (encoding gut -specific esterase) promoter, ts lethal phenotype of the mutant was rescued. These results suggest that Ce-
vps45 gene products may function in membrane trafficking from Golgi's apparatus to lysosome in mainly intestinal cells where lysosome is highly developed, and dysfunction of the lysosome resulting from Ce-
vps45 mutation causes growth arrest and larval lethality in C elegans .
tm246 mutant fed with Rhodamine-Dextran showed granules loaded with bright red fluorescence in the intestinal cells, even though blue fluorescence was amorphous, suggesting that the endocytosis was almost normal in the mutant. We speculate that the lysosomal function may be necessary for processing of neutrition uptaken into intestinal cells. To identify the functional domain for membrane trafficking into the lysosome in intestine, or the plasma membrane in neuron, chimeric experiments between Ce-
vps45 and
unc-18 are in progress.