title: Selective cell-surface labeling of the molecular motor protein prestin.
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.
authors.affiliation: Department of Bioengineering, Rice University, Houston, TX 77251, USA.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: McGuire
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Silberg
authors.forename: Fred A
authors.initials: FA
authors.lastname: Pereira
authors.forename: Robert M
authors.initials: RM
authors.lastname: Raphael
mesh_terms: D000595:Amino Acid Sequence; D027321:Anion Transport Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation; D002462:Cell Membrane / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D000076988:Sulfate Transporters
chemical_list: D027321:Anion Transport Proteins; D011993:Recombinant Fusion Proteins; C498719:SLC26A5 protein, human; D000076988:Sulfate Transporters; D001710:Biotin; D017479:Receptors, Platelet-Derived Growth Factor