Name: measuring plasma membrane protein endocytic rates by reversible biotinylation
Uploaded: 2009-12-23T16:00:00
Description: Watch this Scientific Journal Video about Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation at JoVE.com

This work was funded by NIH grant #DA15169 to H.E.M.

Procedure Overview:
Using this approach, cell surface proteins are covalently labeled with biotin on available extracellular lysine residues using a membrane impermeant, disulfide-coupled biotinylation reagent (sulfo-NHS-SS-biotin) under trafficking restrictive conditions (i.e. low temperature) (see Fig. 1 for illustration). One set of cells is shifted to trafficking permissive conditions (37&deg;C) and biotinylated proteins internalize. The other set of cells are kept at low temperature as c...

<ol>
	<li>Holton, K. L., Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+distinct+endocytic+signals+dictate+constitutive+and+PKC-regulated+neurotransmitter+transporter+internalization.">Nonclassical distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization.</a> Nat Neurosci. 8, 881-888 (2005).</li><li>Boudanova, E., Navaroli, D. M., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+decreases+in+dopamine+transporter+surface+expression+are+protein+kinase+C-independent.">Amphetamine-induced decreases in dopamine transporter surface expression are protein kinase C-independent.</a> Neuropharmacology. 54, 605-612 (2008).</li><li>Boudanova, E., Navaroli, D. M., Stevens, Z., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+transporter+endocytic+determinants:+carboxy+terminal+residues+critical+for+basal+and+PKC-stimulated+internalization.">Dopamine transporter endocytic determinants: carboxy terminal residues critical for basal and PKC-stimulated internalization.</a> Mol Cell Neurosci. 39, 211-217 (2008).</li><li>Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopamine+transporter+constitutively+internalizes+and+recycles+in+a+protein+kinase+C-regulated+manner+in+stably+transfected+PC12+cell+lines.">The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines.</a> J Biol Chem. 278, 22168-22174 (2003).</li></ol>

Common problems: The most common problem that arises in these experiments is poor strip efficiency. The efficiency of the strip is critical in being able to interpret the results. Unless the strip was highly efficient, it is not possible to conclude that any biotinylated proteins in the internalization lanes were, in fact, internalized from the surface. Strips &ge;90% efficiency are optimal, and we discard any results if the strip falls below this level. An example of a poor strip is shown in Figure 3. Note t...

measuring plasma membrane protein endocytic rates by reversible biotinylation

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.

Watch this Scientific Journal Video about Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation at JoVE.com

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

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