Comparison of human INSIG1 and the two yeast proteins Nsg1p and Nsg2p. Protein sequences of these three proteins were compared by T-Coffee at EMBNET. Macboxshade software was used for graphic depiction. The black sections are identities shared by all three proteins. Dark gray are identities shared by any two, and light gray are similarities.

NSG overexpression stabilized Hmg2p-GFP (A) or full-length Hmg2p (B). (A) Log-phase cultures of cells expressing Hmg2p-GFP from the TDH3 promoter were subjected to flow cytometry to evaluate steady-state levels of Hmg2p-GFP fluorescence in the presence of empty vector (‘control'), TDH3-expressed Nsg1p, Nsg2 and Nsg1p-3HA, or the control dominant-negative hemi-Hrd1p. Cells were incubated with no drug, zaragozic acid (ZA) or lovastatin (LOVA) as indicated. Horizontal axis is log fluorescence in arbitrary units, and vertical axis is the number of cells per fluorescent channel. All x-axis in a column are identically aligned to facilitate visual comparison between strains. (B) Log-phase cultures of cells expressing 1myc-Hmg2p from the TDH3 promoter and the indicated proteins were treated with cycloheximide (25 μg/ml) followed by immunoblotting at the indicated times.

Overexpressed NSGs did not stabilize ER degradation substrates CPY^* (A), 6myc-Hmg2p-GFP (B) or Sec61-2 (C). (A) Cells expressing 1myc-Hmg2p (top row) or CPY^* (bottom row), along with empty vector (‘control'), TDH3-driven NSG1 and NSG2, or dominant-negative hemi-HRD1 were treated with cycloheximide for the indicated times and subjected to immunoblotting. (B) Log-phase cultures of cells coexpressing 6myc-Hmg2p-GFP with no protein (‘control'), Nsg1p, Nsg2p or hemi-Hrd1p were treated with cycloheximide and assayed for cellular fluorescence at the indicated times after treatment. A leftward shift indicates a loss of fluorescence due to HRD-dependent degradation. (C) Lack of effect of NSG2 (top rectangle) or NSG1 (bottom plates) overexpression on temperature-sensitive growth of sec61-2 mutants. Top panel: the sec61-2 strain harboring empty vector (‘control'), a plasmid expressing TDH3-driven Nsg2p, TDH3-driven hemi-Hrd1p or wild-type Sec61p were tested for growth by dilution assay on solid medium at the permissive 30°C temperature, or the nonpermissive 35°C temperature. Suspensions were serially diluted five-fold and identical samples of each dilution were spotted on agar and allowed to grow. Note the suppression by the general dominant-negative hemi-Hrd1p is as good as that provided by the complementing the SEC61 plasmid. Bottom circles: duplicate sec61-2 strains harboring vector, plasmids expressing TDH3-driven Nsg1p or wild-type Sec61p in the indicated sectors were placed on agar and allowed to grow at 30 or 35°C.

NSG1 stabilized Hmg2p in a regulation-deficient cod1Δ null. cod1Δ null mutant cells without (top panel) or with (middle panel) overexpressed NSG1 were subjected to cycloheximide treatment (50 μg/ml; light gray) or no drug (black) and incubated for 4 h to assess degradation of the Hmg2p-GFP reporter in this mutant background. Separate control cultures were incubated for 4 h with lovastatin (25 μg/ml; dark gray) to confirm the lack of Hmg2p-GFP regulation, shown by the unchanging steady-state levels. The bottom panel shows an otherwise identical wild-type (wt) strain overexpressing NSG1 subjected to the same experiment for comparison of the extent of NSG stabilization. All three panels have aligned horizontal axes to facilitate visual comparison.

NSG1 functions naturally to limit HRD-dependent, regulated degradation of native Hmg2p. (A) Removal of NSG1 allowed regulated degradation of native Hmg2p. Log-phase cultures of strains expressing native levels of 1myc-Hmg2p from its native promoter with the indicated NSG genotypes (wt, nsg1 null, double null, nsg2 null) were treated for 0 or 4 h with cycloheximide (50 μg/ml) in the presence or absence of lovastatin (25 mg/ml; 4L), and subjected to immunoblotting for myc-Hmg2p. (B) Strains with the indicated NSG and HRD1 genotypes were subjected to the same protocol as in (A) to evaluate HRD dependence of regulated degradation that occurs in the absence of NSGs. (C) Removal of NSGs results in lower levels of native Hmg2p enzyme activity. Strains expressing only native levels of Hmg2p as the sole source of HMGR, with the indicated NSG phenotypes, were compared for sensitivity to lovastatin growth inhibition. Low-density cultures (OD∼0.02) were grown for 49 h in the presence of the indicated concentrations of lovastatin, read for OD₆₀₀, and normalized to the no-drug culture for each genotype. The nsg null was approximately three times more sensitive to lovastatin.

Overexpression of Hmg2p transmembrane domain caused a functional loss of Nsg activity. Strains expressing the native-promoter myc-Hmg2p with the indicated overexpression plasmids (control is empty vector) were compared in cycloheximide chases as in Figures 5 and 6 for regulated stability of native-level myc-Hmg2p. Note that the only myc-tagged protein in these strains is the native promoter expressed myc-Hmg2p.

Direct assays of Nsg1p–Hmg2p interaction. (A) Co-precipitation of myc-Hmg2p by Nsg1p-3HA. Nondenaturing detergent lysates of microsomes from cells coexpressing the two tagged proteins or the individual molecules were immunoprecipitated with anti-HA beads as described in Materials and methods. Samples of the supernatants after precipitation, or protein captured by the beads were resolved by SDS–PAGE and immunoblotted for the Nsg1p-3HA or myc-Hmg2p as indicated. (B, C) Blue native PAGE (BN-PAGE) electrophoresis of Nsg1p-3HA and myc-Hmg2p. Strains expressing myc-Hmg2p alone, and strains coexpressing Nsg1p-3HA with full-length myc-Hmg2p or soluble Hmg2p catalytic domain were lysed and subjected to BN-PAGE followed by immunoblotting. Panel B (‘anti-HA') shows the mobility of Nsg1p-3HA in the presence of coexpressed myc-Hmg2p or the soluble catalytic domain. Panel C (‘anti-myc') shows the mobility of myc-Hmg2p in the presence of empty vector or Nsg1p-3HA. To generate the B and C panels, the same nitrocellulose was first probed for HA, then stripped as described in Materials and methods (‘protein degradation and immunoblotting'), and then reprobed for myc. (D) Immunoblotting of identical samples of each of the three lysates to show total levels of Nsg1p-3HA and myc-Hmg2p.

In vitro assays of Nsg1p action on Hmg2p. (A) Effect of coexpressed Nsg1p on limited protection assay of lumenally tagged myc_L-Hmg2p. Microsomes of otherwise identical strains expressing the lumenally tagged mycL-Hmg2p with or without added Nsg1p were incubated with trypsin for the indicated times, followed by immunoblotting for the lumenal myc tag. (B, C) In vitro ubiquitination of Hmg2p. In vitro ubiquitination of myc-Hmg2p was evaluated by preparing microsomes of various assay strains, and incubation of the microsomes with cytosol with (UBC7) or without (Δ) Ubc7p, followed by lysis, immunoprecipitation of Hmg2p-GFP and immunoblotting for ubiquitin or GFP as indicated. (B) Hrd1p dependence of in vitro Hmg2p-GFP ubiquitination. Microsomes with Hrd1p (‘+'), no Hrd1p (‘Δ') or dominant-negative C399S Hrd1p were compared in ubiquitination assay. (C) Effect of Nsg1p on in vitro ubiquitination of Hmg2p by Hrd1p. Otherwise identical, strains with or without TDH3-expressed Nsg1p were compared. (D) Hrd1p-3HA crosslinking to Hmg2p-GFP in the presence and absence of Nsg1p. Microsomes from strain coexpressing Hrd1p-3HA and Hmg2p-GFP, with (‘NSG1') or without (‘vector') Nsg1p-3HA, were prepared as in the ubiquitination assay, treated with crosslinker DSP at the indicated concentrations for 1 h, quenched and subject to anti-GFP immunoprecitipation and immunoblotting for GFP, or HA. Note that significant Nsg1p is co-precipitated in the absence of crosslinker.