A genome-wide visual screen reveals a role for sphingolipids and ergosterol in cell surface delivery in yeast.
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.
Proszynski
Tomasz J
TJ
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
Klemm
Robin W
RW
Gravert
Maike
M
Hsu
Peggy P
PP
Gloor
Yvonne
Y
Wagner
Jan
J
Kozak
Karol
K
Grabner
Hannes
H
Walzer
Karen
K
Bagnat
Michel
M
Simons
Kai
K
Walch-Solimena
Christiane
C
D001692:Biological Transport / Q000235:genetics / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004875:Ergosterol / Q000096:biosynthesis*; D015723:Gene Library; D005800:Genes, Fungal / Q000235:genetics*; D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D050506:Mutant Chimeric Proteins / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013107:Sphingolipids / Q000096:biosynthesis*; D022161:Transport Vesicles / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism*
D002135:Calcium-Binding Proteins; C490024:FUS1 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; C091625:MID2 protein, S cerevisiae; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D050506:Mutant Chimeric Proteins; D029701:Saccharomyces cerevisiae Proteins; D013107:Sphingolipids; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D004875:Ergosterol