The C-terminus of prestin influences nonlinear capacitance and plasma membrane targeting.
Prestin is a unique molecular-motor protein expressed in the lateral plasma membrane of outer hair cells (OHC) in the organ of Corti of the mammalian cochlea. It is thought that prestin undergoes conformational changes driven by the cell's membrane potential. The resulting alterations in OHC-length are assumed to constitute the cochlear amplifier. Prestin is a member of the anion solute carrier family 26 (SCL26A), but it is different from other family members in its unique function of voltage-driven motility. Because the C-terminus is the least conserved region in the family, we investigated its influence with a series of deletion, point and chimeric mutants. The function and cellular expression of mutants were examined in a heterologous expression system by measurement of nonlinear capacitance (NLC) and immunofluorescence. Each mutant produced a unique mixture of patterns of cell morphologies, which were classified as to the location of prestin within the cell. The data from deletion mutants (Del516, Del525, Del630, Del590, Del709, Del719) revealed that nearly the full length (>708 amino acids) of the protein was required for normal prestin expression and function. Since most deletion mutations eliminated plasma membrane targeting, chimeric proteins were constructed by fusing prestin, at amino acid 515 or 644, with the homologous portion of the C-terminus from the two most closely related SLC26A members, pendrin and putative anion exchanger 1. These chimeric proteins were again improperly (but differently) targeted than simple truncation mutants, and all lacked functional phenotype. When two of the potential basolateral membrane-targeting motifs were mutated (Y520A/Y526A), incomplete plasma membrane expression was seen. We also show that some double point mutations (V499G/Y501H) fully express in the plasma membrane but lack NLC. These non-charged amino acids may have unrevealed important roles in prestin's function. Together, these data suggest that certain specific sequences and individual amino acids in the C-terminus are necessary for correct cellular distribution and function.
Auditory Physiology Laboratory, Department of Communication Sciences and Disorders, Northwestern University, Evanston, IL 60208 USA. jzh215@northwestern.edu
Jing
J
Zheng
Guo-Guang
GG
Du
Keiji
K
Matsuda
Alex
A
Orem
Sal
S
Aguiñaga
Levente
L
Deák
Enrique
E
Navarrete
Laird D
LD
Madison
Peter
P
Dallos
D000818:Animals; D027321:Anion Transport Proteins / Q000378:metabolism; D017920:Antiporters / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D025802:Electric Capacitance; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D005849:Gerbillinae; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008954:Models, Biological; D020409:Molecular Motor Proteins; D016297:Mutagenesis, Site-Directed; D017711:Nonlinear Dynamics; D010641:Phenotype; D017354:Point Mutation; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D000076988:Sulfate Transporters
D027321:Anion Transport Proteins; D017920:Antiporters; D020409:Molecular Motor Proteins; C498720:Pres protein, mouse; D011506:Proteins; C494849:Slc26a4 protein, mouse; C466900:Slc26a6 protein, mouse; D000076988:Sulfate Transporters