Title

Measuring plasma membrane protein endocytic rates by reversible biotinylation

PubMed ID

20032927

UMMS Affiliation

Department of Psychiatry; Department of Biochemistry and Molecular Biology

Date

12-25-2009

Document Type

Article

Subjects

Biotinylation; Cell Membrane; Endocytosis; Membrane Proteins

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps. Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling. Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression. The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins. In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured. For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands. However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate. In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate. This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Rights and Permissions

Citation: J Vis Exp. 2009 Dec 23;(34). pii: 1669. doi: 10.3791/1669. Link to article on publisher's site

Related Resources

Link to Article in PubMed