QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 27 November 2007
doi: 10.1242/jcs.011247

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shapira, I. Articles by Bar-Nun, S. Search for Related Content PubMed PubMed Citation Articles by Shapira, I. Articles by Bar-Nun, S. Social Bookmarking                         What's this?

Distinguishing between retention signals and degrons acting in ERAD

Ilana Shapira¹, Dana Charuvi^1,*, Yechiel Elkabetz^1,, Koret Hirschberg² and Shoshana Bar-Nun^1,

¹ Department of Biochemistry, George S. Wise Faculty of Life Sciences, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
² Department of Pathology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

View larger version (39K): [in this window] [in a new window]   Fig. 1. The µstpCys motif prevents secretion of TPO and targets it to ERAD. (A) Schematic presentation of the Myc-tagged TPO vectors. (B) COS-7 or HEK293T cells transfected with vectors encoding Myc-tagged TPO-µstpCys (Cys) or TPO-µstpSer (Ser) were pulse-labeled with [35S]methionine-[35S]cysteine and chased for the indicated time. Cells and medium were separated, cells were lysed and substrates were immunoprecipitated (IP) from lysed cells or medium by an antibody against Myc. Immunoprecipitates were resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([35S]). (C) The graph, representative of five independent experiments, illustrates the amounts of TPO-µstpCys (Cys; open symbols) or TPO-µstpSer (Ser; filled symbols) remaining in HEK293T cells (circles), recovered from medium (squares) or their sum (triangles). Amounts estimated by densitometry of autoradiograms in B were calculated as a percentage of their levels at the end of the pulse (100%). (D) HEK293T cells transfected with a vector encoding Myc-tagged TPO-µstpCys were pulse-labeled with [35S]methionine-[35S]cysteine, chased for the indicated time with (+) or without (–) ALLN and MG-132 and analyzed as described in B. (E) The graph, representative of five independent experiments, illustrates the amounts of TPO-µstpCys remaining in untreated (open symbols) or ALLN- and MG-132-treated (+ Inhib; filled symbols) HEK293T cells. The amounts detected in cells (circles), recovered from medium (squares) or their sum (triangles) were estimated by densitometry of autoradiogram D, calculated as a percentage of their levels at the end of the pulse (100%), and the half-life values (see text) were calculated.

View larger version (42K): [in this window] [in a new window]   Fig. 2. The µstpCys element prevents secretion of ssYFP and targets it to ERAD. (A) Schematic presentation of the Myc-tagged ssYFP vectors. (B) Hela cells expressing a vector encoding Myc-tagged ssYFP (myc), or HEK293 cell vectors encoding ssYFP-µstpCys (Cys) or ssYFP-µstpSer (Ser), were incubated with (+) or without (–) ALLN and MG-132, pulse-labeled with [35S]methionine-[35S]cysteine and chased for the indicated time with (+) or without (–) ALLN and MG-132. Cells and medium were separated, cells were lysed and substrates were immunoprecipitated (IP) from lysed cells or medium by antibodies against GFP or Myc. Immunoprecipitates were resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([35S]). ssYFP-µstpCys without N-glycan (*), resulting from de-glycosylation or inefficient glycosylation. ssYFP-µstpSer with high-mannose (arrow) or complex (arrowhead) N-glycans are indicated. (C) The graphs, representative of three independent experiments, illustrate the amounts of the indicated ssYFP fusion proteins remaining in untreated (open symbols) or ALLN- and MG-132-treated (+ Inhib; filled symbols) cells. The amounts detected in cells (circles) or recovered from medium (squares) were estimated by densitometry of autoradiograms in B, calculated as a percentage of their levels at the end of the pulse (100%), and the half-life values (see text) were calculated.

View larger version (88K): [in this window] [in a new window]   Fig. 3. The µstpCys element retains ssYFP in the ER. COS-7 cells were transfected with vectors encoding Myc-tagged ssYFP (myc), ssYFP-µstpCys (Cys) or ssYFP-µstpSer (Ser), together with galactosyl transferase-CFP (GalT-CFP). YFP (left panels) and CFP (middle panels) were visualized by confocal fluorescence microscopy individually or as merged images (right panels). Bar, 5 µm.

View larger version (19K): [in this window] [in a new window]   Fig. 4. The µstpCys element, acting as a portable ERAD degron, does not serve as the ubiquitin conjugation site. HEK293T cells were transfected with vectors encoding (A) the wild-type (WT) version of the Myc-tagged TPO-µstpCys or (B) the TPO-µstpCysKR mutant (KR), in which the only K residue in µstp was replaced by arginine. Cells were pulse-labeled with [35S]methionine-[35S]cysteine and chased for the indicated time with (+) or without (–) ALLN and MG-132. Substrates were immunoprecipitated (IP) from lysed cells by an antibody against Myc, immunoprecipitates were resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([35S]). (C) The graph, representative of three independent experiments, illustrates the amounts of TPO-µstpCys (WT; circles) or TPO-µstpCysKR (KR; triangles) remaining in untreated (open symbols) or ALLN- and MG-132-treated (+ Inhib; filled symbols) HEK293T cells. The amounts estimated by densitometry of autoradiograms A and B were calculated as a percentage of their levels at the end of the pulse (100%), and the half-life values (see text) were calculated.

View larger version (25K): [in this window] [in a new window]   Fig. 5. The µstpCys N-glycan decelerates degradation of TPO. HEK293T were transfected with an empty vector (mock), or with vectors encoding Myc-tagged TPO-µstpCys (WT), TPO-µstpCysKR (KR) or TPO-µstpCysNQ (NQ). In the NQ mutant, the only N residue in the µstp motif was replaced by Q, abolishing the µstp glycosylation site. (A) Cells were lysed, samples of cell lysate (10%) were collected and substrates were immunoprecipitated (IP) from the remaining 90% of the lysate by an antibody against Myc. Immunoprecipitates were treated with (+) or without (–) endo H, and immunoprecipitates and cell lysates were resolved by SDS-PAGE and immunoblotted (IB) with an antibody against µstp. Fully glycosylated WT and KR, under-glycosylated NQ and endo-H-treated de-glycosylated substrates are indicated. (B) HEK293T transfected with an empty vector (mock) or with vectors encoding Myc-tagged TPO-µstpCys (WT) or TPO-µstpCysNQ (NQ) were pulse-labeled with [35S]methionine-[35S]cysteine, chased for the indicated time, lysed and substrates were immunoprecipitated (IP) by an antibody against Myc. Immunoprecipitates were resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([35S]; upper panels) to monitor degradation and then immunoblotted (IB) with an antibody against µstp (lower panels) to estimate steady-state levels. (C) The graph, representative of three independent experiments, illustrates the amounts of TPO-µstpCys (WT; circles) or TPO-µstpCysNQ (NQ; triangles) remaining in HEK293T cells. The amounts estimated by densitometry of autoradiograms in B were calculated as a percentage of their levels at the end of the pulse (100%), and half-life values (see text) were calculated.

View larger version (43K): [in this window] [in a new window]   Fig. 6. Hampered secretion and accelerated degradation of the ssYFP-µstpNQ mutants. (A) HEK293T cells were transfected with an empty vector (mock), with vectors encoding Myc-tagged wild-type versions of ssYFP-µstpCys (Cys) or ssYFP-µstpSer (Ser), or the NQ mutants of ssYFP-µstpCys (CysNQ) or ssYFP-µstpSer (SerNQ). In the NQ mutants, the N residue in the µstp was replaced by a Q residue, abolishing the only glycosylation site in the chimera. Cells were lysed, substrates were immunoprecipitated (IP) by an antibody against Myc, immunoprecipitates were treated with (+) or without (–) endo H, resolved by SDS-PAGE and immunoblotted (IB) with an antibody against Myc. Fully glycosylated WT, unglycosylated NQ and endo-H-treated de-glycosylated substrates are indicated. (B) HEK293T cells expressing ssYFP-µstpCysNQ (CysNQ) or ssYFP-µstpSerNQ (SerNQ) were preincubated for 1 hour and pulse-labeled with [35S]methionine-[35S]cysteine and chased for the indicated time with (+) or without (–) ALLN and MG-132. Cells were lysed, substrates were immunoprecipitated (IP) by an antibody against Myc, resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([35S]). (C) The graph, representative of three independent experiments, illustrates the amounts of ssYFP-µstpCysNQ (CysNQ; circles) or ssYFP-µstpSerNQ (SerNQ; squares) remaining in untreated (open symbols) or ALLN- and MG-132-treated (+ Inhib; filled symbols) cells. The amounts estimated by densitometry of autoradiograms in B were calculated as a percentage of the levels of the NQ mutants at the end of the pulse (100%), and half-life values (see text) were calculated. (D) Hela cells were transfected with an empty vector (mock), with vectors encoding Myc-tagged wild-type versions of ssYFP-µstpCys (Cys) or ssYFP-µstpSer (Ser), or the NQ mutants of ssYFP-µstpCys (CysNQ) or ssYFP-µstpSer (SerNQ). Medium was collected 40 hours post-transfection, and the cells were lysed. Substrates were immunoprecipitated (IP) from the medium by an antibody against Myc, and cell lysates (10%) and immunoprecipitates from the medium were resolved by SDS-PAGE and immunoblotted (IB) with an antibody against Myc. Substrates with complex (arrowhead) or high-mannose (arrow) N-glycans and unglycosylated NQ are indicated. (E) COS-7 cells were transfected with a combination of vectors encoding Myc-tagged ssYFP-µstpSerNQ mutant (SerNQ) and galactosyl transferase-CFP (GalT-CFP). YFP (left panel) and CFP (middle panel) were visualized by confocal fluorescence microscopy individually or as merged images (right panels). Bar, 5 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 7. Tunicamycin hampers secretion and accelerates degradation of the ssYFP-µstp fusion proteins. (A) HEK293 cells expressing a vector encoding ssYFP-µstpCys (Cys) were preincubated for 1 hour with tunicamycin. Cells were pulse-labeled with [35S]methionine-[35S]cysteine and chased for the indicated time with tunicamycin together (+) or without (–) ALLN and MG-132. Cells and medium were separated, cells were lysed and ssYFP-µstpCys was immunoprecipitated (IP) from lysed cells or medium by an antibody against Myc. (B) The graph, representative of three independent experiments, illustrates the amounts of ssYFP-µstpCys remaining in untreated (open symbols) or ALLN- and MG-132-treated (+ Inhib; filled symbols) cells. The amounts recovered from medium (M; squares) or detected in cells as combined unglycosylated and glycosylated forms (C; circles) or as unglycosylated forms (C ung; triangles) were estimated by densitometry of autoradiogram A, calculated as a percentage of the ssYFP-µstpCys level at the end of the pulse (100%), and half-life values (see text) were calculated. (C) HEK293T cells expressing a vector encoding ssYFP-µstpCysNQ (CysNQ) were preincubated for 1 hour and chased with (+) or without (–) tunicamycin (Tm). Cells were pulse-labeled with [35S]methionine-[35S]cysteine and chased for the indicated time with or without tunicamycin together (+) or without (–) ALLN and MG-132. ssYFP-µstpCysNQ was immunoprecipitated (IP) from lysed cells by an antibody against Myc. (D) HEK293 cells expressing ssYFP-µstpSer (Ser) were preincubated for 1 hour and chased with (+) or without (–) tunicamycin (Tm). Cells were pulse-labeled with [35S]methionine-[35S]cysteine and chased for the indicated time with or without tunicamycin, together (+) or without (–) ALLN and MG-132. Cells and medium were separated, cells were lysed and substrates were immunoprecipitated (IP) from lysed cells (upper panel) or medium (lower panel) by an antibody against Myc. Immunoprecipitates were resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([35S]). The unglycosylated and the residual glycosylated substrates are indicated. ssYFP-µstpSer with high-mannose (arrow) or complex (arrowhead) N-glycans are indicated. (E) The graphs, representative of three independent experiments, illustrate the amounts of ssYFP-µstpSer remaining in untreated (open symbols) or ALLN- and MG-132-treated (+ Inhib; filled symbols) cells (upper graph) or recovered from medium (lower graph). Glycosylated forms from untreated cells (gly; circles) or unglycosylated (Tm ung; triangles) and glycosylated (Tm gly; squares) forms from tunicamycin-treated cells were estimated by densitometry of autoradiogram D, calculated as a percentage of the level of ssYFP-µstpSer at the end of the pulse (100%), and half-life values (see text) were calculated.

View larger version (51K): [in this window] [in a new window]   Fig. 8. Hampered secretion and accelerated degradation of the ssYFP-µstpSA mutants. (A) HEK293T were transfected with an empty vector (mock), with vectors encoding Myc-tagged wild-type versions of ssYFP-µstpCys (Cys) or ssYFP-µstpSer (Ser), the NQ mutants of ssYFP-µstpCys (CysNQ) or ssYFP-µstpSer (SerNQ) or the SA mutants of ssYFP-µstpCys (CysSA) or ssYFP-µstpSer (SerSA). In the SA mutants, the S residue in µstp was replaced by an alanine residue, abolishing the only glycosylation site in the chimera. The cells were lysed, substrates were immunoprecipitated (IP) by an antibody against Myc, immunoprecipitates were treated with (+) or without (–) endo H, resolved by SDS-PAGE and immunoblotted (IB) with an antibody against Myc. Fully glycosylated WT, unglycosylated SA or NQ and endo-H-treated de-glycosylated substrates are indicated. Asterisk, anti-Myc antibody heavy chain. (B) HEK293T cells expressing ssYFP-µstpCysSA (CysSA) or ssYFP-µstpSerSA (SerSA) were preincubated for 1 hour, pulse-labeled with [35S]methionine-[35S]cysteine and chased for the indicated time with (+) or without (–) ALLN and MG-132. The cells were lysed, substrates were immunoprecipitated (IP) by an antibody against Myc, resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([35S]). (C) The graph, representative of three independent experiments, illustrates the amounts of ssYFP-µstpCysSA (CysSA; circles) or ssYFP-µstpSerSA (SerSA; triangles) remaining in untreated (open symbols) or ALLN- and MG-132-treated (+Inhib; filled symbols) cells. The amounts estimated by densitometry of autoradiograms in B were calculated as a percentage of the levels of the SA mutants at the end of the pulse (100%), and half-life values (see text) were calculated. (D) Hela cells were transfected with an empty vector (mock), with vectors encoding Myc-tagged wild-type versions of ssYFP-µstpCys (Cys) or ssYFP-µstpSer (Ser), or the SA mutants of ssYFP-µstpCys (CysSA) or ssYFP-µstpSer (SerSA). Medium was collected 40 hours post-transfection, cells were lysed, and substrates were immunoprecipitated (IP) from lysed cells and medium by an antibody against Myc. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted (IB) with an antibody against Myc. Substrates with complex (arrowhead) or high-mannose (arrow) N-glycans and unglycosylated SA are indicated. (E) COS-7 cells were transfected with a combination of vectors encoding Myc-tagged ssYFP-µstpSerSA mutant (SerSA) and galactosyl transferase-CFP (GalT-CFP). YFP (left panel) and CFP (middle panel) were visualized by confocal fluorescence microscopy individually or as merged images (right panels). Bar, 5 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter