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Bio Protoc. 2016 Feb 20;6(4). pii: e1739.

VAMP8-3xHA Uptake Assay in HeLa Cells.

Author information

1
Département d'anatomie et de biologie cellulaire, Faculté de Médecine et des Sciences de la Santé. Université de Sherbrooke, Sherbrooke, Canada.
2
Division of Biological Sciences, University of California, San Diego, USA.

Abstract

Transmembrane proteins are rarely exclusively localized to a specific vesicle or an organelle. Most transmembrane proteins undergo complicated trafficking routes. Thus, transmembrane proteins are under constant flux, and at steady state, found on a variety of vesicles or organelles. This characteristic makes the study of their trafficking routes complex, since at any given moment, different molecules are often being trafficked in opposing directions. Pulse-chase experiments can temporally track a specific pool of a transmembrane protein of interest, allowing for the kinetic description of its trafficking route. This type of technique has been used extensively to follow a large array of plasma membrane localized proteins (Diril et al., 2006; Jean et al., 2010). Here, we describe a method that allows the study of VAMP8 trafficking from the plasma membrane to endolysosomal compartments. This method was used to describe a role for MTMR13 and RAB21 in the regulation of VAMP8 trafficking to endolysosomes (Jean et al., 2015).

PMID:
28251172
PMCID:
PMC5328665

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