Sec61p-independent degradation of the tail-anchored ER membrane protein Ubc6p - PubMed (original) (raw)

Sec61p-independent degradation of the tail-anchored ER membrane protein Ubc6p

J Walter et al. EMBO J. 2001.

Abstract

Tail-anchored proteins are distinct from other membrane proteins as they are thought to insert into the endoplasmic reticulum (ER) membrane independently of Sec61p translocation pores. These pores not only mediate import but are also assumed to catalyze export of proteins in a process called ER-associated protein degradation (ERAD). In order to examine the Sec61p dependence of the export of tail-anchored proteins, we analyzed the degradation pathway of a tail-anchored ER membrane protein, the ubiquitin-conjugating enzyme 6 (Ubc6p). In contrast to other ubiquitin conjugating enzymes (Ubcs), Ubc6p is naturally short-lived. Its proteolysis is mediated specifically by the unique Ubc6p tail region. Degradation further requires the activity of Cue1p-assembled Ubc7p, and its own catalytic site cysteine. However, it occurs independently of the other ERAD components Ubc1p, Hrd1p/Der3p, Hrd3p and Der1p. In contrast to other natural ERAD substrates, proteasomal mutants accumulate a membrane-bound degradation intermediate of Ubc6p. Most interestingly, mutations in SEC61 do not reduce the turnover of full-length Ubc6p nor cause a detectable accumulation of degradation intermediates. These data are in accordance with a model in which tail-anchored proteins can be extracted from membranes independently of Sec61p.

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Figures

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Fig. 1. Ubc6p degradation depends on Ubc7p and Cue1p, but noton Ubc1p. (A) Immunoblots loaded with equal amounts of isolated membranes from the strains indicated were probed with αUbc6p antibody. (B) The stability of Ubc6p was analyzed in cycloheximide decay experiments of the same strains: after abrogation of protein translation by the addition of cycloheximide, aliquots were taken at the indicated time points, and membranes were prepared and probed for the amount of Ubc6p. (C) Cycloheximide decay experiment in a strain depleted of UBC1. The asterisk marks Sec61p, which served as a loading control.

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Fig. 2. Ubc6p degradation is mediated by its tail domain. The stability of Ubc4p, Ubc6p–TM and Ubc4ptail was tested in cycloheximide decay experiments and analyzed by immunoblotting. Experiments were conducted as in Figure 1B, except that in the upper two panels total cell lysates were probed. Ubc4p was tested in a wild-type strain. Ubc6p deleted for its transmembrane anchor (Ubc6p–TM) and the Ubc4p–Ubc6ptail fusion (Ubc4ptail) were expressed from a low-copy plasmid in a Δubc6 strain. The asterisk marks Sec61p, which served as a loading control.

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Fig. 3. Ubc6p degradation depends on polyubiquitylation, catalytically active Ubc7p and its own activity. Cycloheximide decay experiments conducted as in Figure 1B and analyzed by immunoblotting. (A and B) The genomic UBC7 and UBC6 genes were mutated in their active site cysteine resulting in _ubc7_ser and _ubc6_ser, which were tested for the stability of Ubc6p and Ubc6pser, respectively. (C) The HA-tagged Ubc6p (Ubc6pha) was expressed from a low-copy plasmid in a wild-type strain and in the _ubc6_ser strain. The asterisk marks Sec61p, which served as a loading control. (D) The dependence of Ubc6p on polyubiquitylation was tested by the co-expression of mutant ubiquitinK48R. (E) Δubc6 cells were transformed with high-copy plasmids coding for Ubc6p, Ubc6pser or an empty vector. Microsome preparations from equal amounts of cells were immunoprecipitated with anti-Ubc6p antibody, and western blots were analyzed with an anti-ubiquitin antibody. The Ubc6p immunoprecipitation was controlled by blotting aliquots for Ubc6p (data not shown).

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Fig. 4. Proteasomal mutants stabilize Ubc6p and cause the accumulation of a degradation intermediate. (A) Cycloheximide decay experiment of the proteasomal mutant strain pre1-1, conducted as in Figure 1B and analyzed by immunoblotting. A fragment accumulated, which reacted with the αUbc6p antibody. (B) Western blot of membrane preparations from the strains indicated. (C) Western blot of total cell lysates from the strains indicated. (D) Membranes from the pre1-1 strain were extracted with agents that strip peripheral proteins from the membranes, and with detergent, as indicated. Equal amounts of supernatant (sn) and pellet (p) were blotted for Ubc6p. The asterisk marks Sec61p, which served as a loading control.

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Fig. 5. Ubc6p degradation does not depend on Hrd1p/Der3p, Hrd3p or Der1p. (A) The immunoblots show cycloheximide decay experiments, conducted as in Figure 1B, of Δhrd1/der3, Δhrd3 and Δder1 deletion strains and of the corresponding wild-type strains. All strains expressed the prc1-1 allele, which codes for the classical ERAD substrate CPY*. It served as a control for the stabilizing effects on ERAD. The asterisk (*) marks Sec61p, which served as a loading control. (B) Quantification of the luminescence of the Ubc6p signals in an experiment as in (A).

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Fig. 6. Sec61p function in Ubc6p degradation. (A and B) Cycloheximide decay experiment conducted in sec61 mutant strains and the corresponding wild type. Membranes were immunoblotted and probed for the indicated proteins. The asterisk marks Sec61p, which served as a loading control. The experiments in (A) and (B) were conducted at 25°C. CPY* demonstrates the translocation defects of these strains. The lower band reacting with the αCPY antibody indicates the non-glycosylated precursor, which results from an impaired import. The amount of Ubc6p was determined using a luminescence imager. The relative intensity is given as a percentage of the value at the zero time point. (C) The indicated yeast strains treated with cycloheximide for 3 h at 25°C. Aliquots before and after cycloheximide treatment were taken and membranes immunoblotted for Ubc6p degradation intermediates. (D) Radioactive pulse–chase experiment of the same strains as in (A) after a 1 h pre-shift to 37°C. Total cell lysates were immuno precipitated for Ubc6p. (E) Quantification of experiments as shown in (D). Three independent experiments were averaged and the standard deviation was calculated and is indicated by error bars (values for prc1-1 are 100%; 45.1 ± 1.9%; 17.5 ± 3.6%; 12.0 ± 1.7%; and for prc1-1, sec61-2 are 100%; 43.4 ± 10.8%; 21.3 ± 6.1%; 14.5 ± 1.7%).

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