A Novel Assay to Identify the Trafficking Proteins That Bind to Specific Vesicle Populations

Overview

Journal Curr Protoc Cell Biol

Specialty Cell Biology

Date 2015 Dec 2

PMID 26621371

Citations 15

Authors

Marvin Bentley

Gary Banker

Affiliations

Soon will be listed here.

Abstract

Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin binding domain (FRB)-tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking. We describe two versions of the assay: a general protocol for use in cells with a typical microtubule-organizing center and a specialized protocol designed to detect protein-vesicle interactions in cultured neurons. We have successfully used this assay to identify kinesins and Rabs that bind to a variety of different vesicle populations. In principle, this assay could be used to investigate interactions between any category of vesicle trafficking proteins and any vesicle population that can be specifically labeled.

Citing Articles

Kinesin Regulation in the Proximal Axon is Essential for Dendrite-selective Transport.

Mendoza C, Plowinske C, Montgomery A, Quinones G, Banker G, Bentley M Mol Biol Cell. 2024; 35(6):ar81.

PMID: 38598291 PMC: 11238084. DOI: 10.1091/mbc.E23-11-0457.

Polarized transport requires AP-1-mediated recruitment of KIF13A and KIF13B at the trans-Golgi.

Montgomery A, Mendoza C, Garbouchian A, Quinones G, Bentley M Mol Biol Cell. 2024; 35(5):ar61.

PMID: 38446634 PMC: 11151104. DOI: 10.1091/mbc.E23-10-0401.

A Chemically Inducible Organelle Rerouting Assay to Probe Primary Cilium Assembly, Maintenance, and Disassembly in Cultured Cells.

Turan F, Ercan M, Firat-Karalar E Methods Mol Biol. 2023; 2725:55-78.

PMID: 37856017 DOI: 10.1007/978-1-0716-3507-0_3.

KAP is the neuronal organelle adaptor for Kinesin-2 KIF3AB and KIF3AC.

Garbouchian A, Montgomery A, Gilbert S, Bentley M Mol Biol Cell. 2022; 33(14):ar133.

PMID: 36200888 PMC: 9727798. DOI: 10.1091/mbc.E22-08-0336.

Axonal transport of Hrs is activity dependent and facilitates synaptic vesicle protein degradation.

Birdsall V, Kirwan K, Zhu M, Imoto Y, Wilson S, Watanabe S Life Sci Alliance. 2022; 5(10).

PMID: 35636965 PMC: 9152131. DOI: 10.26508/lsa.202000745.

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