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Mating-based Overexpression Library Screening in Yeast
Published on: July 6, 2018
An overexpression screen in Saccharomyces cerevisiae identifies novel genes that affect endocytic protein trafficking
Henning Arlt1, Angela Perz, Christian Ungermann
1Department of Biology/Chemistry, Biochemistry section, University of Osnabrück, Osnabrück 49076, Germany.
Abstract:
A large number of proteins involved in the biogenesis of yeast endosomes and vacuoles have been identified based on screens that scored for inactivation of proteins. Such screens may, however, miss important regulators of the pathway. Here, we present a visual screen in which we examined the effects on vacuole morphology if any of the 6153 yeast open reading frames was overexpressed. Using a progressive screening procedure, we could identify a total of 53 genes. Among the most striking endosomal proteins are the CORVET/HOPS subunits Vps3, Vps18 and Vps39 and the putative tethering inhibitor Ivy1. Furthermore, six endosomal sorting complex related to transport (ESCRT) proteins led to altered vacuole morphology if overproduced. Among the novel proteins, we identify Yer128w as an endosomal protein that interacts with the AAA-ATPase Vps4, and therefore named it Vfa1 (Vps Four-Associated 1). We present evidence on the possible role of these novel proteins in trafficking to the vacuole. Our data provide novel insights into the regulation of protein trafficking.
Insights
This study used a visual screen to identify novel yeast genes regulating endosome and vacuole biogenesis. Overexpression of 53 genes, including CORVET/HOPS and ESCRT components, revealed new insights into protein trafficking pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Yeast Genetics
Background:
- Traditional screens for yeast endosome and vacuole biogenesis proteins often miss key regulators.
- Visual screening offers an alternative approach to identify novel components involved in these cellular pathways.
Purpose of the Study:
- To identify novel genes regulating yeast endosome and vacuole morphology through a visual overexpression screen.
- To characterize the roles of newly identified proteins in protein trafficking to the vacuole.
Main Methods:
- A large-scale visual screen of 6153 yeast open reading frames for altered vacuole morphology upon overexpression.
- Progressive screening procedures to identify significant gene candidates.
- Interaction studies to characterize novel protein functions, such as Vfa1 with Vps4.
Main Results:
- Identified 53 genes influencing vacuole morphology, including known CORVET/HOPS subunits (Vps3, Vps18, Vps39) and ESCRT proteins.
- Discovered novel endosomal proteins, including Yer128w (renamed Vfa1), which interacts with the AAA-ATPase Vps4.
- Provided evidence for the involvement of novel proteins in vacuolar trafficking.
Conclusions:
- Overexpression screening is a powerful tool for discovering novel regulators of endosome and vacuole biogenesis.
- The identified genes and proteins, particularly Vfa1, offer new insights into the complex mechanisms of protein trafficking and vacuolar organization in yeast.
