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.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-10192005-030729 |
| Date | 20 March 2006 |
| Creators | Youker, Robert Thomas |
| Contributors | Jeffrey L Brodsky, Raymond A Frizzell, Jeffrey D Hildebrand, Karen M Arndt, John D Hempel |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-10192005-030729/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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