Prestin topology: localization of protein epitopes in relation to the plasma membrane.
Computer modeling of the outer hair cell (OHC) motor protein prestin produces ambiguous results regarding transmembrane regions and localization of its termini. To determine the location of prestin's N- and C-termini, we created prestin constructs with synthetic epitopes located immediately upstream or downstream of prestin. The spatial distribution of these epitopes was studied in prestin-transfected cells using immunofluorescence. In permeabilized cells, antibodies label the plasma membrane of 30% of the cells, reflecting transfec- tion efficiency. Under non-permeabilizing conditions, the few labeled cells also displayed a lack of plasma membrane integrity. These data suggest that prestin's N-and C-termini are cytoplasmic. Furthermore, prestin staining in OHCs was observed only under permeabilizing conditions. These results implicate prestin's N- and C-termini as portions that may interact with other cytoplasmic proteins. A model of prestin membrane topology is also considered based on the results.
Zheng
J
J
Auditory Physiology Laboratory (The Hugh Knowles Center), Department of Neurobiology and Physiology and Communication Sciences and Disorders, Northwestern University, Frances Searle Building, 2299 North Campus Drive, Evanston, IL 60208, USA.
Long
K B
KB
Shen
W
W
Madison
L D
LD
Dallos
P
P
D000818:Animals; D000918:Antibody Specificity / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005849:Gerbillinae; D049452:Green Fluorescent Proteins; D018072:Hair Cells, Auditory, Outer / Q000737:chemistry* / Q000648:ultrastructure; D006309:Hearing / Q000502:physiology; D007202:Indicators and Reagents / Q000032:analysis; D008164:Luminescent Proteins / Q000032:analysis; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D017434:Protein Structure, Tertiary / Q000502:physiology; D011506:Proteins / Q000737:chemistry*; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
D018076:DNA, Complementary; D000939:Epitopes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011506:Proteins; D049452:Green Fluorescent Proteins