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Recently synthesized proteins are sorted at the trans-Golgi network into specialized routes for exocytosis. Surprisingly little is known about the underlying molecular machinery. Here, we present a visual screen to search for proteins involved in cargo sorting and vesicle formation. We expressed a GFP-tagged plasma membrane protein in the yeast deletion library and identified mutants with altered marker localization. This screen revealed a requirement of several enzymes regulating the synthesis of sphingolipids and ergosterol in the correct and efficient delivery of the marker protein to the cell surface. Additionally, we identified mutants regulating the actin cytoskeleton (Rvs161p and Vrp1p), known membrane traffic regulators (Kes1p and Chs5p), and several unknown genes. This visual screening method can now be used for different cargo proteins to search in a genome-wide fashion for machinery involved in post-Golgi sorting.

Original publication

DOI

10.1073/pnas.0509107102

Type

Journal article

Journal

Proc Natl Acad Sci U S A

Publication Date

13/12/2005

Volume

102

Pages

17981 - 17986

Keywords

Biological Transport, Calcium-Binding Proteins, DNA Mutational Analysis, Ergosterol, Gene Library, Genes, Fungal, Green Fluorescent Proteins, Intracellular Signaling Peptides and Proteins, Membrane Glycoproteins, Membrane Proteins, Mutant Chimeric Proteins, Phenotype, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sphingolipids, Transport Vesicles, Vesicular Transport Proteins, trans-Golgi Network