Usa1 Functions as a Scaffold of the HRD-Ubiquitin Ligase Sabine C. Horn, Jennifer Hanna, Christian Hirsch, Corinna Volkwein, Anja Schütz, Udo Heinemann, Thomas Sommer, Ernst Jarosch  Molecular Cell  Volume 36, Issue 5, Pages 782-793 (December 2009) DOI: 10.1016/j.molcel.2009.10.015 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Usa1 Is Required for Basic Functions of the HRD-Ligase (A) Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10-fold dilutions and incubated for 3 days at the indicated temperatures. Where indicated, 2 mg/ml tunicamycin (+ TM) was added to the medium to affirm the hac1-knockout. (B) Quantification of pulse-chase experiments to determine the degradation of Sec61-2 (left panel) and 6xmyc-Hmg2 (right panel). The relevant genotype of the strains used is given. Error bars represent the standard deviation of mean of at least three experiments. Molecular Cell 2009 36, 782-793DOI: (10.1016/j.molcel.2009.10.015) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Usa1 Is an Integral Component of the HRD-Ligase (A) Left panel: Microsomal preparations from logarithmically growing cells expressing Usa1-myc were solubilized with NP-40 and subjected to immunoprecipitation with antibodies against the myc-epitope. Samples were analyzed by SDS-PAGE followed by immunoblotting using specific antibodies. Right panel: A scheme representing the results obtained in the left panel. (B) Microsomal NP-40 lysates derived from strains that expressed myc epitope-tagged Hrd3 and were deleted for the indicated genes were subjected to immunoprecipitation and analyzed as in (A). Molecular Cell 2009 36, 782-793DOI: (10.1016/j.molcel.2009.10.015) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 The N-Terminal Region of Usa1 Binds to a Cytoplasmic Part of Hrd1 (A) Schematic drawing depicting the predicted topologies of Hrd1 and Usa1. Numbers refer to the position of amino acids. The locations of transmembrane segments (TM), the ubiquitin-like (UBL-), and RING-finger domains are given. (B) Usa1-myc yeast cells transformed with constructs for the expression of either the N-terminal (Usa11–535) or the C-terminal (Usa1548–838) region of Usa1 were solubilized with digitonin-buffer and subjected to HA- or myc-immunoprecipitation. After separation by SDS-PAGE, the samples were analyzed by immunoblotting. (C) Yeast cells expressing either Hrd1-HA or Hrd11–517-HA were solubilized with digitonin-buffer and subjected to HA-immunoprecipitation. After separation by SDS-PAGE, the samples were analyzed by immunoblotting. (D) Left panel: The N terminus of Usa1 (Usa11–535, theoretical mass 64 kDa) and the C terminus of Hrd1 (Hrd1325–551, theoretical mass 29 kDa) were individually expressed as N-His7 fusions in E. coli and purified. Subsequently, the proteins were subjected to gel filtration either individually or after mixing and incubation at 4°C. Apparent molecular masses were calculated on the basis of the elution profile of the following substances: blue dextran 2000 (2 MDa, void volume), thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa). Right panel: The eluted proteins in the indicated fractions were analyzed by SDS-PAGE and Coomassie staining. Numbers refer to the masses of standard proteins in kilodaltons. Molecular Cell 2009 36, 782-793DOI: (10.1016/j.molcel.2009.10.015) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Oligomerization of Hrd1 Relies on Usa1 (A) Solubilized microsomes from diploid cells expressing either wild-type (WT; lanes 1 and 6), or WT and HA-tagged Hrd3 (lanes 2 and 7), or WT and HA-tagged Hrd1 (lanes 4 and 9) were subjected to immunoprecipitation with HA-antibodies under nondenaturing conditions. A mixture of haploid strains expressing Hrd3 or HA-Hrd3 (lanes 3 and 8) and WT or Hrd1-HA (lanes 5 and 10), respectively, was used as controls. Asterisks denote cross-reacting material. Note that, for unknown reasons, the Hrd1 antiserum only poorly reacts with Hrd1-HA in the input material (Gauss et al., 2006b). (B) Yeast cells expressing either WT or an active-site cysteine mutant of Hrd1 (Hrd1C/S) were transformed with a low-copy plasmid expressing HA-tagged variants of either protein (Hrd1-HA and Hrd1C/S-HA, respectively). Membranes were prepared, solubilized under nondenaturing conditions, and subjected to immunoprecipitation with HA antibodies. (C) Strains deleted for the given genes or expressing a catalytically inactive variant of Ubc7 (Ubc7C/S) were transformed with a Hrd1-HA expressing low copy plasmid. After solubilization of prepared microsomes, Hrd1-HA was immunoprecipitated, and the samples were analyzed by immunoblotting. Molecular Cell 2009 36, 782-793DOI: (10.1016/j.molcel.2009.10.015) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 A Usa1 Mutant Defective for the Degradation of Membrane Proteins (A) Turnover of CPY∗ (left panel) or 6xmyc-Hmg2 (right panel) in the indicated yeast strains was determined in pulse-chase experiments. A quantification of at least 3 individual experiments and error bars of the standard deviation are given. (B) Yeast cells of the indicated genotype were lysed, and 6xmyc-Hmg2 was precipitated with anti-myc antibodies. The precipitates were analyzed by SDS-PAGE and immunoblotting. rpt4R refers to a yeast strain with reduced proteasomal activity due to a catalytically inactive version of the AAA-ATPase Rpt4. (C) Cells of the indicated genotype expressing 6xmyc-Hmg2 and Hrd1C/S-HA were lysed under nondenaturing conditions. After precipitation with HA-antibodies, the associated material was analyzed by SDS-PAGE followed by immunoblotting. (D) Microsomes prepared from cells expressing the indicated Usa1 variants were solubilized under nondenaturing conditions and subjected to immunoprecipitation. Samples were then analyzed by SDS-PAGE and immunoblotting. (E) Immunoprecipitation of Hrd1-myc from membrane preparations lysed under nondenaturing conditions. Molecular Cell 2009 36, 782-793DOI: (10.1016/j.molcel.2009.10.015) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Distinct Functions of the Usa1 N and C Termini (A) WT or usa1-deleted cells that expressed Hrd1 along with Hrd1-HA and the indicated versions of Usa1 were lysed in digitonin-buffer and subjected to HA-immunoprecipitation. (B) Yeast cells expressing the indicated Usa1 constructs were incubated with cycloheximide and at the given time points, samples were removed, lysed, and analyzed by SDS-PAGE and immunoblotting. Immunoblotting with Sec61 antibodies served as a loading control. Ubc6-turnover was monitored to exclude unspecific effects on ubiquitin-proteasome mediated degradation. (C) Cycloheximide-decay assay to monitor the degradation of CPY∗ as described in (B). (D) Microsomes were prepared from yeast cells coexpressing HA-tagged variants of Hrd1 and WT or truncated Hrd1 versions. After lysis with nondenaturing detergent, immunoprecipitation was performed with HA antibodies and the samples were analyzed by immunoblotting. Asterisks denote a Usa1-fragment that was supposedly generated by unspecific proteolysis during extract preparation. (E) Cycloheximide-decay assay to monitor the turnover of 6xmyc-Hmg2 in Δhrd1 cells that expressed the indicated Hrd1 variants as described in (B). (F) CPY∗ degradation was measured by the quantification of pulse-chase experiments in Δhrd1 cells expressing the given Hrd1 versions. Mean values of at least 2 individual experiments are shown. Molecular Cell 2009 36, 782-793DOI: (10.1016/j.molcel.2009.10.015) Copyright © 2009 Elsevier Inc. Terms and Conditions