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A novel immunodetection screen for vacuolar defects identifies a unique allele of VPS35 in S. cerevisiae

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Abstract

The late endosome and vacuole of yeast Saccharomyces cerevisiae are functionally equivalent to the mammalian late endosome and lysosome. The late endosome is the convergence point of the biosynthetic and endocytic trafficking to the vacuole. Here, we describe a novel immunodetection screen to isolate mutants defective in trafficking the soluble hydrolase carboxypeptidase Y (CPY) at the late endosome to vacuole interface (env mutants). Mutants exhibit vacuolar morphology and endocytosis defects as assayed by electron, fluorescent, and nomarski microscopy. In biochemical assays, they internally accumulate p2CPY in a dense membrane compartment lacking vacuolar properties yet display normal secretion phenotypes. The results suggest vacuolar morphology and function defects that are exclusively at the late endosome/vacuole interface. env mutants define five complementation groups. The first gene of the collection to be cloned, ENV1 is allelic to VPS35 whose established function is in retrograde trafficking from late endosome to trans-Golgi network (TGN). Microscopic, biochemical, and growth analyses establish that env1 is distinct from other alleles of VPS35 in vacuolar morphology, growth characteristics, and internal accumulation of p2CPY. Our results indicate that ENV genes may define new gene functions at the late endosome to vacuole interface.

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Acknowledgments

We are grateful to G. Payne and B. Horazdovsky for continuous support, guidance, and plasmid reagents. We thank T. Stevens for original gift of pro-CPY antibody and the Emr laboratory for anti-Vps35 antibodies. This work was supported by NIH-MBRS-SCORE grant # GM63119-02 to EG. MKT and OC were partially supported by the NIH-MBRS-SCORE grant. MP, CF, and DS were partially supported by NIH-MBRS, Howard Hughes, and Boeing, respectively.

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  1. Department of Biological Sciences, California State University at Long Beach, 1250 Bellflower Blvd, Long Beach, CA, 90840, USA

    M. Kathleen Takahashi, Christopher Frost, Ken Oyadomari, Marcos Pinho, Dyna Sao, Onyi Chima-Okereke & Editte Gharakhanian

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  1. M. Kathleen Takahashi
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  2. Christopher Frost
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Correspondence to Editte Gharakhanian.

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Takahashi, M.K., Frost, C., Oyadomari, K. et al. A novel immunodetection screen for vacuolar defects identifies a unique allele of VPS35 in S. cerevisiae . Mol Cell Biochem 311, 121–136 (2008). https://doi.org/10.1007/s11010-008-9703-y

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