A microscopy-based kinetic analysis of yeast vacuolar protein sorting

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Abstract

Saccharomyces cerevisiae is amenable to studying membrane traffic by live-cell fluorescence microscopy. We used this system to explore two aspects of cargo protein traffic through prevacuolar endosome (PVE) compartments to the vacuole. First, at what point during Golgi maturation does a biosynthetic vacuolar cargo depart from the maturing cisternae? To address this question, we modified a regulatable fluorescent secretory cargo by adding a vacuolar targeting signal. Traffic of the vacuolar cargo requires the GGA clathrin adaptors, which arrive during the early-to-late Golgi transition. Accordingly, the vacuolar cargo begins to exit the Golgi near the midpoint of maturation, significantly before exit of a secretory cargo. Second, how are cargoes delivered from PVE compartments to the vacuole? To address this question, we tracked biosynthetic and endocytic cargoes after they had accumulated in PVE compartments. The results suggest that stable PVE compartments repeatedly deliver material to the vacuole by a kiss-and-run mechanism.

Data availability

Newly created plasmids will be archived with Addgene. Yeast strains are freely available upon request to any interested researcher.

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Jason C Casler

Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-9742-9978
Benjamin S Glick

Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, United States

For correspondence
bsglick@uchicago.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-7921-1374

Funding

National Institutes of Health (R01 GM104010)

Benjamin S Glick

National Institutes of Health (T32 GM007183)

Jason C Casler

National Institutes of Health (P30 CA014599)

Benjamin S Glick

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.