A study of the C-terminus in prestin by designing cleavable constructs

The membrane protein prestin plays a central role in the mammalian auditory system by enabling Outer Hair Cells (OHCs) in the cochlea to actively respond to electrical signals. Prestin belongs to the SLC26A family of membrane proteins, which all have a large cytosolic terminus containing a conserved region known as the STAS domain (Sulfate Transporters and Anti-Sigma factor antagonist), whose function re- mains unknown. In this work, we engineered prestin by inserting three types of short peptides, flexible, rigid and cleavable linkers, into different positions in the C- terminus and tested prestin’s funtionality, self-interactions, and lateral mobility in the membrane. First, we inserted short peptides between the last trans-membrane domain and the STAS domain. We found that the prestin’s functionality is inhibited, and the self-interaction is significantly decreased. The FRAP data revealed that the mobility of prestin is altered by inserting different types of linkers before the STAS domain. Second, we inserted cleavable linkers in the disordered region within the STAS domain. The data revealed that prestins inserted with linkers between 596aa and 597aa, and between 620aa and 621aa, can successfully locate to the membrane and retain NLC function. Next, by using advanced fluorescence microscopy and im- munoprecipitation, we discovered that prestin is confined by a binding site lcoated between the 597aa and the 620aa. Together, these data suggest that the interface between the C-terminus of prestin, STAS domain, and the last TMD is crucial to prestin’s function, self-interactions, and mobility, and that a binding site locates in the disordered region of STAS domain that influence prestin’s lateral mobility.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Engineering

Grantor

Rice University

Year dc:date.issued

2016

Author dc:creator

• Guo, Jing

Advisor dc:contributor.advisor

• Raphael, Robert M

• Prestin
• STAS
• TEV
• Microscopy

Statement dc:rights

• Copyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1911/95968

OAI identifier oai:identifier

oai:repository.rice.edu:1911/95968