Analysis of the Oligomeric Structure of the Motor Protein Prestin

Jing Zheng,Guo-Guang Du,Charles T Anderson,Jacob P Keller,Alex Orem,Peter Dallos,Maryann Cheatham

doi:10.1074/jbc.m513854200

Abstract

Prestin, a member of the solute carrier family 26, is expressed in the basolateral membrane of outer hair cells. This protein provides the molecular basis for outer hair cell somatic electromotility, which is crucial for the frequency selectivity and sensitivity of mammalian hearing. It has long been known that there are abundantly expressed approximately 11-nM protein particles present in the basolateral membrane. These particles were hypothesized to be the motor proteins that drive electromotility. Because the calculated size of a prestin monomer is too small to form an approximately 11-nM particle, the possibility of prestin oligomerization was examined. We investigated possible quaternary structures of prestin by lithium dodecyl sulfate-PAGE, perfluoro-octanoate-PAGE, a membrane-based yeast two-hybrid system, and chemical cross-linking experiments. Prestin, obtained from different host or native cells, is resistant to dissociation by lithium dodecyl sulfate and behaves as a stable oligomer on lithium dodecyl sulfate-PAGE. In the membrane-based yeast two-hybrid system, homo-oligomeric interactions between prestin-bait/prestin-prey suggest that prestin molecules can associate with each other. Chemical cross-linking experiments, perfluoro-octanoate-PAGE/Western blot, and affinity purification experiments all indicate that prestin exists as a higher order oligomer, such as a tetramer, in prestin-expressing yeast, mammalian cell lines and native outer hair cells. Our data from experiments using hydrophobic and hydrophilic reducing reagents suggest that the prestin dimer is connected by a disulfide bond embedded in the prestin hydrophobic core. This stable dimer may act as the building block for producing the higher order oligomers that form the approximately 11-nM particles in the outer hair cell's basolateral membrane.

Highlights

Prestin, the outer hair cells (OHCs) motor protein [8], is located at the same location where the 11-nM protein particles are found, i.e. in the lateral membrane of OHCs (9 –11)
When prestin is heterologously expressed in several mammalian cell lines, the prestinexpressing cells demonstrate all of the characteristics that are unique to OHCs; that is, voltage-driven non-linear capacitance and shape changes [8], electro-mechanical reciprocality [12, 13], and mechanical force generation with constant amplitude and phase up to a stimulating frequency of at least 20 kHz [12]
2 The abbreviations used are: OHC, outer hair cell; LDS, lithium dodecyl sulfate; PFO, perfluoro-octanoate; SLC26, solute carrier 26; anti-C-mPres, a polyclonal antibody against C terminus of mouse prestin protein; KO, knockout; PBS, phosphate-buffered saline; C-tag prestin, gerbil prestin tagged at the C terminus with the hexahistidine and V5 epitope; CAT, chloramphenicol acetyltransferase; DTT, dithiothreitol; NEXT-PAGE, New Electrophoresis X’PRESS Technology PAGE; BMH, bismaleimidohexane; BS3, bis(sulfosuccinimidyl) suberate; SD-L, SC medium without leucine; SD-LT, SC medium without leucine and tryptophan; SD-LTH, SC medium without leucine, tryptophan, and histidine; Nub, the N-terminal domain of ubiquitin; Cub, the C-terminal domain of ubiquitin; NubG, the mutant form of Nub; X-gal, 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside

Summary

Introduction

The OHC motor protein [8], is located at the same location where the 11-nM protein particles are found, i.e. in the lateral membrane of OHCs (9 –11). When prestin is heterologously expressed in several mammalian cell lines, the prestinexpressing cells demonstrate all of the characteristics that are unique to OHCs; that is, voltage-driven non-linear capacitance and shape changes [8], electro-mechanical reciprocality [12, 13], and mechanical force generation with constant amplitude and phase up to a stimulating frequency of at least 20 kHz [12]. Based on the average volume per mass ratio of known proteins, a simple calculation shows that it is highly unlikely that one prestin subunit would inhabit an area as large as that occupied by the 11-nM particles. This raises the question of whether prestin exists as an oligomer in the plasma membrane. Results demonstrate that prestin can form higher order oligomers, probably tetramers, both in OHCs and in heterologous expression systems

Objectives

Results

Conclusion