New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy

Despite the importance of clathrin-mediated endocytosis (CME) for cell biology, it is unclear if all components of the machinery have been discovered and many regulatory aspects remain poorly understood. Here, using Saccharomyces cerevisiae and a fluorescence microscopy screening approach we identif...

Full description

Saved in:

Bibliographic Details
Published in:	Genetics (Austin) Vol. 201; no. 3; pp. 1061 - 1070
Main Authors:	Farrell, Kristen B, Grossman, Caitlin, Di Pietro, Santiago M
Format:	Journal Article
Language:	English
Published:	United States Genetics Society of America 01-11-2015
Subjects:	Clathrin - metabolism Endocytosis Fluorescence Green Fluorescent Proteins Investigations Microscopy Microscopy, Fluorescence - methods Peptide Library Recombinant Fusion Proteins Saccharomyces cerevisiae Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae - metabolism Saccharomyces cerevisiae Proteins - genetics Saccharomyces cerevisiae Proteins - metabolism Yeast endocytosis clathrin yeast machinery
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	Despite the importance of clathrin-mediated endocytosis (CME) for cell biology, it is unclear if all components of the machinery have been discovered and many regulatory aspects remain poorly understood. Here, using Saccharomyces cerevisiae and a fluorescence microscopy screening approach we identify previously unknown regulatory factors of the endocytic machinery. We further studied the top scoring protein identified in the screen, Ubx3, a member of the conserved ubiquitin regulatory X (UBX) protein family. In vivo and in vitro approaches demonstrate that Ubx3 is a new coat component. Ubx3-GFP has typical endocytic coat protein dynamics with a patch lifetime of 45 ± 3 sec. Ubx3 contains a W-box that mediates physical interaction with clathrin and Ubx3-GFP patch lifetime depends on clathrin. Deletion of the UBX3 gene caused defects in the uptake of Lucifer Yellow and the methionine transporter Mup1 demonstrating that Ubx3 is needed for efficient endocytosis. Further, the UBX domain is required both for localization and function of Ubx3 at endocytic sites. Mechanistically, Ubx3 regulates dynamics and patch lifetime of the early arriving protein Ede1 but not later arriving coat proteins or actin assembly. Conversely, Ede1 regulates the patch lifetime of Ubx3. Ubx3 likely regulates CME via the AAA-ATPase Cdc48, a ubiquitin-editing complex. Our results uncovered new components of the CME machinery that regulate this fundamental process.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1943-2631 0016-6731 1943-2631
DOI:	10.1534/genetics.115.180729