title: Rapid turnover of stereocilia membrane proteins: evidence from the trafficking and mobility of plasma membrane Ca(2+)-ATPase 2.
abstract: We studied the spatial distribution, mobility, and trafficking of plasma membrane Ca2+ATPase-2 (PMCA2), a protein enriched in the hair cell apical membrane and essential for hair cell function. Using immunofluorescence, we determined that PMCA2 is enriched in the stereocilia and present at a relatively low concentration in the kinocilium and in the remaining apical membrane. Using an antibody to the extracellular domain of PMCA2 as a probe, we observed that PMCA2 diffuses laterally from the stereocilia membrane and is internalized at the apical cell border maintaining an estimated half-life of residency in the stereocilia of approximately 5-7 h. A computer simulation of our data indicates that PMCA2 has an estimated global diffusion coefficient of 0.01-0.005 microm2/s. Using a green fluorescent protein tag, we observed that PMCA2 is rapidly delivered to the apical cell border from where it diffuses to the entire stereocilia surface. Fluorescence recovery after photobleaching experiments show that approximately 60% of PMCA2 in the stereocilia exhibit high mobility with a diffusion coefficient of 0.1-0.2 microm2/s, whereas the remaining pool represents a relatively immobile fraction. These results suggest that PMCA2 molecules maintain transient interactions with other components of the stereocilia, and the mobile pool of PMCA2 mediates the exchange between the stereocilia and the removal and delivery sites at the periphery of the apical cell surface. This rapid turnover of a major stereocilia membrane protein matches the previously described rapid turnover of proteins of the stereocilia actin core, further demonstrating that these organelles undergo rapid continuous renewal.
authors.affiliation: Laboratory of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: M'hamed
authors.initials: M
authors.lastname: Grati
authors.forename: Mark E
authors.initials: ME
authors.lastname: Schneider
authors.forename: Karen
authors.initials: K
authors.lastname: Lipkow
authors.forename: Emanuel E
authors.initials: EE
authors.lastname: Strehler
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Wenthold
authors.forename: Bechara
authors.initials: B
authors.lastname: Kachar
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002923:Cilia / Q000201:enzymology / Q000378:metabolism*; D004705:Endocytosis; D006198:Hair Cells, Auditory / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D053499:Plasma Membrane Calcium-Transporting ATPases; D051381:Rats; D014018:Tissue Distribution
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D027682:Cation Transport Proteins; D008565:Membrane Proteins; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases