Sequence determinants for regulated degradation of yeast 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein

Abstract

The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway. To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p. When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized. The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation. When amino acid residues 27-54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated. The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes. These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes. Neither determinant was sufficient to impart regulated degradation to Hmg1p. These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.

Figure 1

Cartoon of the chimeric proteins in this study. The ovals represent either the appropriate HMG-R catalytic region (which provides HMG-R enzymatic activity), or the GFP optical reporter protein, as indicated in text. Thick lines represent sequence from Hmg1p; thin lines represent sequence from Hmg2p. The box labeled “M” indicates the presence of the myc epitope tag in the poorly conserved linker region.

Figure 2

The first 26 amino acids of Hmg1p stabilized Hmg2p. (A) Cycloheximide-chase assay of strains expressing either wild-type Hmg2p or Hmg2p with the first 26 amino acids replaced with those from Hmg1p (2–1_1–26). Cycloheximide was added to cultures at 0 h, and samples were lysed immediately or after a 4-h degradation period and then analyzed by immunoblotting with the 9E10 anti-myc antibody. (B) Effect of ZA on the log-phase, steady-state levels of normal Hmg2p or 2–1_1–26. Cultures of strains expressing the normal or chimeric protein were allowed to grow in the absence (−) or presence (ZA) of 25 μg/ml ZA. Cell samples were then lysed and immunoblotted as in panel a. (C) Amino acid sequence comparison of the first 26 amino acids of Hmg1p and Hmg2p. In all figures, the strain used to express a given recombinant or optical reporter can be found in Table 1.

Figure 3

Stabilizing replacement of the first 26 amino acids of Hmg2p-GFP did not affect cellular localization of the protein. (A) Cycloheximide-chase assay of strains expressing Hmg2p-GFP or the stabilized chimera 2–1_1–26-GFP. After addition of cycloheximide at 0 h, lysates were prepared at the indicated times and immunoblotted with anti-GFP antibody. (B) Fluorescence microscopy of living, log-phase cultures of strains expressing Hmg2p-GFP or the stabilized chimera, 2–1_1–26-GFP.

Figure 4

Reduced regulation of Hmg2p by replacement of the first transmembrane span with that from Hmg1p. (A) The regulation of normal Hmg2p and the first transmembrane span chimera of Hmg2p (2–1_27–54) were assayed in a cycloheximide-chase experiment. After addition of cycloheximide, samples of each strain were lysed at 0 h, or after a 4-h incubation in the absence (4) or presence (4L) of 25 μg/ml lovastatin, followed by immunoblotting of all lysates using the 9E10 anti-myc antibody. (B) Amino acid sequence comparison of the amino acids 27–54 of Hmg1p and Hmg2p.

Figure 5

Effect of lovastatin on 2–1_27–24 was less potent than on normal Hmg2p. (A) Effect of drug treatments on the log-phase, steady-state levels of normal Hmg2p or 2–1_27–54. Log-phase cultures of strains expressing the indicated protein were grown in the presence of the indicated doses of lovastatin (Lov), or 25 μg/ml ZA. Levels were determined by immunoblotting with the 9E10 anti-myc antibody after a 4-h growth period. (B) Effect of indicated doses of lovastatin on the log-phase, steady-state levels of Hmg2p-GFP or 2–1_27–54-GFP in strains with identical HMG-R activity. The strains expressing each reporter protein were otherwise isogenic, and each expressed the soluble, nondegraded Hmg2p catalytic domain as the sole source of HMG-R activity. Each strain was grown in the indicated dose of lovastatin (μg/ml) for 3 h and then analyzed by FACS for levels of the fluorescent reporter protein. Horizontal axis represents arbitrary fluorescence units on a log scale. Vertical axis represents cell numbers for a given fluorescence intensity. A total of 10,000 cells for each histogram were analyzed.

Figure 6

Degradation of the poorly regulated 2–1_27–54 is HRD1 dependent. (A) Cycloheximide-chase assay of strains expressing normal Hmg2p or 2–1_27–54 in the presence of a normal HRD1 gene (2–1_27–54) or the hrd1–1 allele (2–1_27–54, hrd1–1). After addition of cycloheximide, lysates were prepared at the indicated times and immunoblotted with the 9E10 anti-myc antibody. (B) FACS histograms of strains expressing the GFP versions of normal Hmg2p or 2–1_27–54 in strains with the HRD1 or hrd1–1 allele. All strains had identical HMG-R catalytic activity supplied by expression of the Hmg2p catalytic domain. Strains were analyzed by FACS directly from early-log-phase cultures. Axes were as described in Figure 5.

Figure 7

Levels of ubiquitination for Hmg2p, 2–1_1–26, and 2–1_27–54 were correlated with their different degradative behaviors. Strains coexpressing an HA-tagged ubiquitin (expressed from the CUP1 promoter) with either normal Hmg2p, 2–1_1–26, or 2–1_27–54 (expressed from the GAPDH promoter) were grown at 30°C to an OD₆₀₀ of 0.2. Expression of the HA-tagged ubiquitin was induced by incubating the cells at 30°C in the presence of 100 μM CuSO₄ for 60 min. ZA was added to a final concentration of 10 μg/ml to the indicated cultures (ZA) for the final 10 min of incubation. Cultures were lysed and the Hmg2p proteins were immunoprecipitated with antibodies raised against Hmg2p. The levels of ubiquitinated Hmg2p were assayed by immunoblotting with the 12CA5 anti-HA antibody (top panel). Immunoprecipitates from strains incubated with (ZA) or without (−) ZA are indicated. The levels of Hmg2p immunoprecipitated were assayed by immunoblotting with the 9E10 anti-myc antibody (bottom panel).

Figure 8

The first 26 amino acids of Hmg1p stabilized the poorly regulated 2–1_27–54. (A) Cycloheximide-chase assays of strains expressing normal Hmg2p, 2–1_27–54, or 2–1_1–54 (which has both the stabilizing and the deregulating replacements). After addition of cycloheximide, lysates were prepared at the indicated times and immunoblotted with the 9E10 anti-myc antibody. (B) The log-phase, steady-state levels of normal Hmg2p, 2–1_27–54, or 2–1_1–54 were assayed in the same strains as in panel a but grown in the presence of either no drug (−), or 25 μg/ml ZA. The levels of Hmg2p in each strain were assayed by immunoblotting with the 9E10 anti-myc antibody after a 3-h growth period in the drugs.

Figure 9

Expression of the poorly regulated 2–1_27–54 had phenotypic consequences. Strains expressing normal Hmg2p, 2–1_1–26, 2–1_27–54, or 2–1_1–54 were tested for lovastatin sensitivity by plating serially diluted cultures on minimal medium plates with (bottom panel) or without (top panel) 200 μg/ml lovastatin, as described. 2–1_27–54 was also tested in an otherwise isogenic hrd1–1 strain.

Figure 10

A segment of the Hmg2p N terminus was sufficient for degradation when used to replace the same region in the Hmg1p transmembrane domain. (A) Cycloheximide-chase assays of strains expressing either normal Hmg2p, 2–1_212–524, or 2–1_1–524. After addition of cycloheximide, lysates were prepared at the indicated times and immunoblotted with the 9E10 anti-myc antibody. (B) Effect of ZA on the log-phase, steady-state levels of the normal Hmg2p or 2–1_212–524. Strains expressing the indicated protein were allowed to grow for 3 h in the absence (−) or presence of 25 μg/ml ZA and then lysed and immunoblotted with the 9E10 anti-myc antibody.