Quantitative Analysis of Endocytosis with Cytoplasmic PHluorin Chimeras

Overview

Journal Traffic

Publisher Wiley

Specialties Biology
Physiology

Date 2010 Jul 15

PMID 20626707

Citations 40

Authors

Derek C Prosser

Karen Whitworth

Beverly Wendland

Affiliations

Soon will be listed here.

Abstract

The pH-sensitive green fluorescent protein (GFP) variant pHluorin is typically fused to the extracellular domain of transmembrane proteins to monitor endocytosis. Here, we have turned pHluorin inside-out, and show that cytoplasmic fusions of pHluorin are effective quantitative reporters for endocytosis and multivesicular body (MVB) sorting. In yeast in particular, fusion of GFP and its variants on the extracellular side of transmembrane proteins can result in perturbed trafficking. In contrast, cytoplasmic fusions are well tolerated, allowing for the quantitative assessment of trafficking of virtually any transmembrane protein. Quenching of degradation-resistant pHluorin in the acidic vacuole permits quantification of extravacuolar cargo proteins at steady-state levels and is compatible with kinetic analysis of endocytosis in live cells.

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