Selective cell-surface labeling of the molecular motor protein prestin

Journal Article · Fri Jun 24 04:00:00 EDT 2011 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/J.BBRC.2011.05.121· OSTI ID:22204973

McGuire, Ryan M. ^[1]; Pereira, Fred A. ^[1]

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1. Department of Bioengineering, Rice University, Houston, TX 77251 (United States)

Highlights: {yields} Trafficking to the plasma membrane is required for prestin function. {yields} Biotin acceptor peptide (BAP) was fused to prestin through a transmembrane domain. {yields} BAP-prestin can be metabolically labeled with biotin in HEK293 cells. {yields} Biotin-BAP-prestin allows for selective imaging of fully trafficked prestin. {yields} The biotin-BAP-prestin displays voltage-sensitive activity. -- Abstract: Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.

• OSTI ID: 22204973
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 1 Vol. 410; ISSN 0006-291X; ISSN BBRCA9
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
AMINO ACIDS
BIOTIN
CYTOPLASM
ELECTROPHYSIOLOGY
FLUORESCENCE
GROWTH FACTORS
LABELLING
PEPTIDES
PROTEIN ENGINEERING
RECEPTORS