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Dissection of Swa2p/auxilin domain r...

Xiao, Jing.

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Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo./
Author:	Xiao, Jing.
Description:	126 p.
Notes:	Adviser: Todd R. Graham.
Contained By:	Dissertation Abstracts International68-12B.
Subject:	Biology, Cell. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3293886
ISBN:	9780549382751

Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo.
Xiao, Jing.

Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo. - 126 p.

Adviser: Todd R. Graham.

Thesis (Ph.D.)--Vanderbilt University, 2007.

The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p contains three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain and a C-terminal J-domain. A Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly.

ISBN: 9780549382751Subjects--Topical Terms:

1017686
Biology, Cell.

Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo.
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020 $a 9780549382751
035 $a (UMI)AAI3293886
035 $a AAI3293886
040 $a UMI $c UMI
100 1 $a Xiao, Jing. $3 1269174
245 1 0 $a Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo.
300 $a 126 p.
500 $a Adviser: Todd R. Graham.
500 $a Source: Dissertation Abstracts International, Volume: 68-12, Section: B, page: 7829.
502 $a Thesis (Ph.D.)--Vanderbilt University, 2007.
520 $a The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p contains three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain and a C-terminal J-domain. A Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly.
520 $a We further examined the influence of the TPR domain on the J-domain stimulation of yHsp70 ATPase. We obtained three surprising results: (1) Swa2p fragment 519-561 (linker) confers a novel HPD-independent J-domain stimulation of yHsp70. (2) GST-(288-668)tpr-j (tpr-linker- j) can robustly stimulate yHsp70 ATPase, but the same mutations renders Swa2p nonfunctional in vivo. (3) GST-(288-668) (TPR-linker-J) does not support stimulation of yHsp70 ATPase in vitro, suggesting the presence of an intra-molecular interaction that is auto-inhibitory. Indeed, the TPR domain interacts in trans with the J-domain. The TPR domain can also mediate interactions with AP1 beta-subunit, Vps1p (yeast dynamin homologue), and Cdc28p (major cyclin-dependent kinase in yeast). These interactions may regulate the co-chaperone activity of Swa2p. Lastly, the association of Swa2p with membranes in a high-speed pellet requires clathrin. A detailed characterization of the fractionation profiles of different Swa2 mutant proteins suggested that clathrin disassembly is required for the disassociation of Swa2p from membranes in the high-speed pellet.
590 $a School code: 0242.
650 4 $a Biology, Cell. $3 1017686
650 4 $a Biology, Molecular. $3 1017719
690 $a 0307
690 $a 0379
710 2 $a Vanderbilt University. $3 1017501
773 0 $t Dissertation Abstracts International $g 68-12B.
790 $a 0242
790 1 0 $a Graham, Todd R., $e advisor
791 $a Ph.D.
792 $a 2007
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3293886