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, Mary Ann Cheatham

*Corresponding author for this work

Research output: Contribution to journalArticle

72 Citations (Scopus)

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 ∼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 ∼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/prestinprey 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 ∼11-nM particles in the outer hair cell's basolateral membrane.

Original languageEnglish (US)
Pages (from-to)19916-19924
Number of pages9
JournalJournal of Biological Chemistry
Volume281
Issue number29
DOIs
StatePublished - Jul 21 2006

Fingerprint

Outer Auditory Hair Cells
Cells
Oligomers
Yeast
Membranes
Two-Hybrid System Techniques
Hybrid systems
Dimers
Proteins
Experiments
Oligomerization
Audition
Cell membranes
Disulfides
Hearing
Purification
Yeasts
Monomers
Western Blotting
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Zheng, J., Du, G. G., Anderson, C. T., Keller, J. P., Orem, A., Dallos, P., & Cheatham, M. A. (2006). Analysis of the oligomeric structure of the motor protein prestin. Journal of Biological Chemistry, 281(29), 19916-19924. https://doi.org/10.1074/jbc.M513854200
Zheng, Jing ; Du, Guo Guang ; Anderson, Charles T. ; Keller, Jacob P. ; Orem, Alex ; Dallos, Peter ; Cheatham, Mary Ann. / Analysis of the oligomeric structure of the motor protein prestin. In: Journal of Biological Chemistry. 2006 ; Vol. 281, No. 29. pp. 19916-19924.
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Zheng, J, Du, GG, Anderson, CT, Keller, JP, Orem, A, Dallos, P & Cheatham, MA 2006, 'Analysis of the oligomeric structure of the motor protein prestin', Journal of Biological Chemistry, vol. 281, no. 29, pp. 19916-19924. https://doi.org/10.1074/jbc.M513854200

Analysis of the oligomeric structure of the motor protein prestin. / Zheng, Jing; Du, Guo Guang; Anderson, Charles T.; Keller, Jacob P.; Orem, Alex; Dallos, Peter; Cheatham, Mary Ann.

In: Journal of Biological Chemistry, Vol. 281, No. 29, 21.07.2006, p. 19916-19924.

Research output: Contribution to journalArticle

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AU - Zheng, Jing

AU - Du, Guo Guang

AU - Anderson, Charles T.

AU - Keller, Jacob P.

AU - Orem, Alex

AU - Dallos, Peter

AU - Cheatham, Mary Ann

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N2 - 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 ∼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 ∼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/prestinprey 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 ∼11-nM particles in the outer hair cell's basolateral membrane.

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Zheng J, Du GG, Anderson CT, Keller JP, Orem A, Dallos P et al. Analysis of the oligomeric structure of the motor protein prestin. Journal of Biological Chemistry. 2006 Jul 21;281(29):19916-19924. https://doi.org/10.1074/jbc.M513854200