The yeast vacuole is functionally and structurally equivalent to the mammalian lysosome. Maintenance of vacuolar function requires correct delivery, modification and retention of proteins. While delivery of resident proteins to their specific organelles is vital for cellular operations, failure to correctly target proteins is correlated with the development of debilitating neurodegenerative and lysosomal storage diseases. In our laboratory, a novel screen for vacuolar trafficking defects at the late-endosome to vacuole has uncovered a unique class of Saccharomyces cerevisiae endosome and vacuole interface ( env) mutants characterized by internal accumulation of carboxy peptidase Y (CPY) exclusively between the endosome and vacuole. Complementation studies of one of these mutants, env1, established that it is a VPS35 allele predicted to encode a truncated Vps35 protein. As a member of retromer, Vps35p binds directly to cargo and facilitates their retrograde transport to the trans-Golgi-network (TGN) from endosomes. Previously reported vps35 mutants secrete p2CPY due to perturbations in retromer function. Our previous studies established that env1 exhibits unique pleotropic phenotypes in comparison to other vps35 alleles. These include fragmented vacuoles growth sensitivity to Hygromycin B and internal p2CPY accumulation. Thus, we hypothesized that the env1 mutant defines a post-endosomal role for Vps35p distinct from all other vps35 mutants and will distribute retromer cargo to the TGN. This hypothesis was tested by exploring the env1 retrograde trafficking state and the delivery and processing of Alkaline Phosphatase (ALP), a vacuolar hydrolase that is not dependent on retrograde trafficking. Sub-cellular fractionation and immunofluorescence microscopy revealed Kex2p- and VpsIOp-TGN localization in env1. Further, immunofluorescence microscopy and Western analysis demonstrated that ALP was properly delivered to the vacuole in env1, but a protion remained unprocessed. Together, these data provide compelling evidence that env1 is unlike other vps35 mutants and does not mislocalize retromer cargo to the vacuole. In addition, the unprocessed vacuolar ALP in env1 suggests an intimate Vps35p involvement in post-endosomal trafficking or processing.
|School:||California State University, Long Beach|
|School Location:||United States -- California|
|Source:||MAI 49/04M, Masters Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Biochemistry|
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