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The Role of Molecular Chaperones in the ER Associated Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator in the Budding Yeast S. cerevisiae

Youker, Robert Thomas (2006) The Role of Molecular Chaperones in the ER Associated Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator in the Budding Yeast S. cerevisiae. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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The requirements of Hsp90 and Hsp70 cytoplasmic chaperone in the proper folding/degradation of an integral membrane protein remain poorly characterized, however it was previously demonstrated that the yeast Hsp70, Ssa1p, chaperone catalyzes the degradation of the misfolded human chloride channel, CFTR. To better define the roles of these chaperones and partner co-chaperones, I characterized the involvement of two Hsp70 co-chaperones, Ydj1p and Hlj1p, in the degradation of CFTR in the budding yeast S. cerevisiae. Mutations in the genes encoding Ydj1p or Hlj1p alone did not affect CFTR degradation, but disruption of both co-chaperones stabilized CFTR. In contrast, the degradation of a soluble misfolded protein (CPY*) was unaffected in an hlj1Δydj1-151 double mutant. Hlj1p stimulated the ATPase activity of Ssa1p and partially rescued the growth defect in a ydj1-151 strain, suggesting that Hlj1p and Ydj1p function redundantly during CFTR degradation. The contribution of Hsp90 to CFTR folding and degradation in mammalian cells has been examined, but disparate results have been obtained. I therefore analyzed CFTR degradation in yeast using a temperature sensitive Hsp90 mutant (Hsp90-G313N) and found that CFTR was degraded faster in the mutant compared to the wildtype. Consistent with this result, highly enriched yeast Hsp90 prevented the aggregation of CFTR's NBD1 domain. In contrast, the degradation of CPY* was unaffected in the Hsp90 mutant. Furthermore, I found no effect on CFTR degradation upon inactivation of the yeast Hsp90 co-chaperones Sba1p, Sti1p, or Sse1p. These results suggest that Hsp90, in the absence of co-chaperones, facilitates CFTR folding, possibly through its interaction with NBD1. Finally, I analyzed the effects of overexpressing two mammalian co-chaperones on CFTR biogenesis in yeast. I observed reduced CFTR degradation upon overexpression of FKBP8 or Bag-3 but did not observe enhanced trafficking of CFTR to the plasma membrane. This result suggests that stabilization per se is not sufficient to promote CFTR exit from the ER.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Youker, Robert
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairBrodsky, Jeffrey Ljbrodsky@pitt.eduJBRODSKY
Committee MemberHildebrand, Jeffrey Djeffh@pitt.eduJEFFH
Committee MemberHempel, John Dhempel@pitt.eduHEMPEL
Committee MemberArndt, Karen Marndt@pitt.eduARNDT
Committee MemberFrizzell, Raymond Afrizzell@pitt.eduFRIZZELL
Date: 20 March 2006
Date Type: Completion
Defense Date: 30 September 2005
Approval Date: 20 March 2006
Submission Date: 19 October 2005
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: ABC Transporter; Conformational Diseases; ER Quality Control; J-protein; Misfolded Membrane Protein; Secretory Pathway
Other ID:, etd-10192005-030729
Date Deposited: 10 Nov 2011 20:03
Last Modified: 15 Nov 2016 13:50


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