spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 14 April 2009
doi: 10.1242/jcs.044255

Journal of Cell Science 122, 1374-1381 (2009)
Published by The Company of Biologists 2009
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kostova, Z.
Right arrow Articles by Weissman, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kostova, Z.
Right arrow Articles by Weissman, A. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

A Ubc7p-binding domain in Cue1p activates ER-associated protein degradation

Zlatka Kostova, Jennifer Mariano, Simone Scholz, Carolin Koenig and Allan M. Weissman*

Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA


Figure 1
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 1. The CUE domain of Cue1p is not necessary for in vivo ERAD. (A) Schematic representation of wild-type and CUE domain mutants of Cue1p with transmembrane (TM) and CUE domains indicated. The LAP sequence within the Cue1p CUE domain (residues 76-78) was mutated as indicated or the entire CUE domain was deleted. (B) 35S pulse-chase analysis of CPY* degradation in {Delta}cue1 cells bearing vector (V), wild-type CUE1, cue1AVA or cue1MFP expressed from low copy plasmids. The graph on the right is quantification of pulse-chase data. (C) Pulse-chase analysis of CPY* in {Delta}cue1 cells expressing vector containing no insert (V), CUE1 or cue1{Delta}CD. The graph on the right is quantification of pulse-chase data. (D) The {Delta}cue1 strain bearing the chromosomal CPY* allele was co-transformed with a plasmid encoding membrane bound CTG* and the indicated wild-type or mutant CUE1-containing vectors. Degradation of CTG* and CPY* was assessed by cycloheximide (CHX) chase and immunoblotting using anti-CPY. Pgk1p (phosphoglycerate kinase) levels were assessed to determine relative protein loading. (E) Degradation of the DOA10 ligase substrate Ste6p*HA in {Delta}cue1 with vector (V), CUE1 or cue1{Delta}CD plasmids was assessed by 35S pulse-chase labeling. Immunoprecipitation was with anti-HA antibody. Graphical representation of the Ste6p*HA degradation is shown on the right. Data are the means and standard deviations of two independent experiments.

 

Figure 2
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 2. The C-terminal region of Cue1p binds Ubc7p and is necessary for ERAD. (A) Schematic representation of GST fusions of Cue1p tested for Ubc7p binding. Shown below for comparison is the C-terminal cytoplasmic domain of gp78 showing the position of the RING finger, CUE domain and Ube2G2-binding region (G2BR). For both, numbering corresponds to positions in the full-length proteins. (B) 35S-labeled in vitro translated Ubc7p was incubated with similar amounts of the indicated GST-Cue1p fusion proteins prebound to glutathione-Sepharose beads (supplementary material Fig. S1). After washing, bound material was resolved by SDS-PAGE. Ten percent of the input used in binding assays is shown. (C) The {Delta}cue1 strain bearing the chromosomal CPY* allele was co-transformed with a plasmid encoding membrane bound CTG* and vectors expressing either wild-type CUE1 or the C-terminal truncation, cue11-173. Degradation of CTG* and CPY* was assessed by cycloheximide (CHX) chase and immunoblotting using anti-CPY. Pgk1p was used as a loading control. A longer exposure of the CTG* immunoblot is shown to show expression and degradation of CTG* in the wild-type strain. (D) 35S pulse-chase analysis of degradation of CPY* in {Delta}cue1 cells expressing vector (V), CUE1 or cue11-173 (vector and CUE1 controls are identical to those in Fig. 1C).

 

Figure 3
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 3. Soluble Cue1p can restore in vivo ERAD and stabilize Ubc7p. (A) Cycloheximide (CHX) chase assays were performed to determine degradation of CPY* and stability of Ubc7pHA in {Delta}cue1{Delta}ubc7 expressing Ubc7pHA together with the indicated regions of Cue1p. A longer exposure of the HA immunoblot is also shown to demonstrate that Ubc7pHA is expressed under all conditions. (B) {Delta}cue1 cells were transformed with plasmids containing no CUE1 gene (V), CUE1 or cue124-203. Degradation of CPY* was assayed by cycloheximide chase analysis at the indicated times. Pgk1p was used as loading control.

 

Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 4. Membrane tethered Ubc7p requires Cue1p to restore ERAD. (A) Schematic representation of soluble (Ubc7pHA) and membrane tethered (TMUbc7pHA) Ubc7p. Ubc7p was C-terminally HA-tagged (black box). Residues 1-44 of Cue1p, containing the N-terminal transmembrane (TM) domain plus ~20 cytosolic residues, were N-terminally fused to Ubc7pHA to generate TMUbc7pHA. (B) {Delta}cue1 cells were transformed with plasmids expressing TMUbc7pHA or Ubc7pHA. Stability of TMUbc7pHA was determined by cycloheximide (CHX) chase. (C) {Delta}ubc7 cells expressing CPY* from the endogenous locus were transformed with empty vector (V) or vectors encoding Ubc7pHA or TMUbc7pHA and degradation of CPY* assessed by 35S pulse-chase. Graphical representation of CPY* degradation is shown on right. Data are the means and s.d. of two independent experiments. (D) {Delta}cue1{Delta}ubc7 cells transformed with plasmids encoding TMUbc7pHA or Ubc7pHA and Cue1p were assessed for CPY* degradation by cycloheximide chase.

 

Figure 5
View larger version (35K):
[in this window]
[in a new window]

  		Fig. 5. The U7BR of Cue1p is necessary and sufficient for ERAD. (A) {Delta}cue1 cells were co-transformed with plasmids encoding the indicated proteins and CPY* degradation monitored by cycloheximide (CHX) chase. Expression of Ubc7pHA and its rapid degradation in cells expressing cue11-173 is apparent on longer exposures (supplementary material Fig. S2). (B) Degradation of CPY* and stability of Ubc7pHA in the double null {Delta}cue1{Delta}ubc7 strain expressing TMUbc7pHA alone or together with the indicated regions of Cue1p. (C) Graph of 35S pulse-chase analysis of CPY* degradation in {Delta}cue1{Delta}ubc7 cells expressing TMUbc7pHA and the indicated forms of Cue1p. Data are the means and s.d. of two independent experiments.

 

Figure 6
View larger version (44K):
[in this window]
[in a new window]

  		Fig. 6. Cue1p151-203 is sufficient to enhance Ubc7p activity in vitro. (A) Equimolar amounts of GST, GST-Ubc7p or GST-UbcH5b were pre-bound to glutathione-Sepharose, and the indicated purified fragments of Cue1p were added to the reaction mix containing ubiquitin and recombinant human E1. For Cue1p25-203, Cue1p25-173 and Cue1p25-{Delta}CD-203 30 pmol was used in each reaction, whereas for Cue1p151-203 240 pmol was used. Following incubation at 30°C, beads were extensively washed and material bound to glutathione-Sepharose was eluted by heating in SDS-PAGE buffer containing β-mercaptoethanol to remove thiolester-linked ubiquitin from the GST fusions, resolved by SDS-PAGE and immunoblotted with anti-ubiquitin. (B) E3-dependent ubiquitylation reactions were performed using GST-Hrd1pC (cytoplasmic domain) pre-bound to glutathione-Sepharose, purified Ubc7p or UbcH5b and 30 pmol of the Cue1p fragments. Samples were treated and analyzed as in A. (C) Reactions were performed essentially as in B, but with increasing amounts of Cue1p151-203. The amount of Cue1p25-203 used was 30 pmols, whereas the amount of Cue1p151-203 ranged from ~60 pmol (lane 3) to 360 pmols (lane 6).

 

Figure 7
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 7. Schematic representation of Ubc7p-Cue1p interaction. Association with the C-terminal U7BR of Cue1p leads to activation of ubiquitin-bound Ubc7p, which, in the presence of an active RING finger domain on the E3 ligase Hrd1p, allows polyubiquitylation of substrates. The CUE domain of Cue1p is dispensable for activation of Ubc7p and ERAD and the transmembrane anchor of Cue1p is similarly not required, at least for the test substrate CPY*. The C-terminal 151-203 amino acids of Cue1p (U7BR), however, mediate the interaction with Ubc7p and are both necessary and sufficient for in vivo ERAD and in vitro polyubiquitylation. In the schematic the asterisk indicates Ubc7~Ub activated by Cue1p. S~Ub4 represents a polyubiquitylated substrate undergoing movement out of the ER through a putative `retrotranslocon'.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2009