HRD gene dependence of endoplasmic reticulum-associated degradation

Abstract

Work from several laboratories has indicated that many different proteins are subject to endoplasmic reticulum (ER) degradation by a common ER-associated machinery. This machinery includes ER membrane proteins Hrd1p/Der3p and Hrd3p and the ER-associated ubiquitin-conjugating enzymes Ubc7p and Ubc6p. The wide variety of substrates for this degradation pathway has led to the reasonable hypothesis that the HRD (Hmg CoA reductase degradation) gene-encoded proteins are generally involved in ER protein degradation in eukaryotes. We have tested this model by directly comparing the HRD dependency of the ER-associated degradation for various ER membrane proteins. Our data indicated that the role of HRD genes in protein degradation, even in this highly defined subset of proteins, can vary from absolute dependence to complete independence. Thus, ER-associated degradation can occur by mechanisms that do not involve Hrd1p or Hrd3p, despite their apparently broad envelope of substrates. These data favor models in which the HRD gene-encoded proteins function as specificity factors, such as ubiquitin ligases, rather than as factors involved in common aspects of ER degradation.

Figure 1

Hmg2p degradation is HRD dependent and UBC6 independent. (A) Cycloheximide–chase assay of strains expressing 1myc-Hmg2p in a wild-type (RHY1611), hrd1Δ (RHY1626), hrd2-1 (RHY1628), hrd3Δ (RHY1631), and ubc7Δ (RHY1633) genetic background. After addition of cycloheximide, lysates were prepared at the indicated times and immunoblotted with the 9E10 anti-myc antibody. (B) Cycloheximide–chase assay of strains expressing 1myc-Hmg2p in a wild-type (RHY1611), ubc6Δ (RHY1723), and ubc7Δ (RHY1633) genetic background. (C) Fluorescence histogram of strains expressing Hmg2p–GFP in a wild-type (RHY871), hrd1Δ (RHY880), ubc7Δ (RHY1056), and hrd1Δubc7Δ (RHY1486) genetic background. Strains were analyzed directly from early log-phase cultures. Each histogram represents 10,000 cells.

Figure 2

Deg1-Hmg2p was completely dependent on UBC7 but only partially dependent on HRD1, HRD3, and UBC6. (A) Cycloheximide–chase assay of strains expressing Deg1-Hmg2p in a wild-type (RHY1610), hrd1Δ (RHY1613), hrd2-1 (RHY1615), hrd3Δ (RHY1617), and ubc7Δ (RHY1619) genetic background. After addition of cycloheximide, lysates were prepared at the indicated times and immunoblotted with the 9E10 anti-myc antibody. An arrow marks the 60-kDa proteolytic fragment seen in hrd2-1 strains. (B) Pulse–chase analysis of the identical strains in A. Cells were pulse-labeled with ³⁵S-Express for 10 min and chased for the indicated times. Deg1-Hmg2p was immunoprecipitated and analyzed by SDS-PAGE and autoradiography. The levels of Deg1-Hmg2p for each time point were determined by densitometric analysis of the autoradiograms. (C) Cycloheximide–chase assay of strains expressing Deg1-Hmg2p in a wild-type (RHY1610), ubc6Δ (RHY1656), and ubc7Δ (RHY1619) genetic background. (D) Fluorescence histogram of strains expressing Deg1-Hmg2p–GFP in a wild-type (RHY1374), hrd1Δ (RHY1575), hrd2-1 (RHY1577), hrd3Δ (RHY1579), and ubc7Δ (RHY1581) genetic background. Strains were analyzed directly from early log-phase cultures. Each histogram represents 10,000 cells.

Figure 3

Deg1-Hmg1p degradation was completely dependent on UBC7 but only partially dependent on the HRD genes and UBC6. (A) Degradation of Deg1-Hmg1p. Results of cycloheximide–chase assay of strains expressing Hmg1p (RHY636) and Deg1-Hmg1p (RHY493) are shown. After addition of cycloheximide, lysates were prepared at the indicated times and immunoblotted with the 9E10 anti-myc antibody. (B) Ubiquitination of Deg1-Hmg1p. Cultures of strains coexpressing an HA-tagged ubiquitin with either Hmg1p (RHY1467) or Deg1-Hmg1p (RHY1460) were lysed, and either Hmg1p or Deg1-Hmg1p was immunoprecipitated with antibodies raised against HMGR. Ubiquitination of the proteins was assayed by immunoblotting with the 12CA5 anti-HA antibody. (C) Cycloheximide–chase assay of strains expressing Deg1-Hmg1p in a wild-type (RHY1948), hrd1Δ (RHY1949), hrd2-1 (RHY2079), hrd3Δ (RHY1950), and ubc7Δ (RHY2096) genetic background. Arrowhead marks the 60-kDa proteolytic fragment seen in hrd2-1 strains. (D) Cycloheximide–chase assay of strains expressing Deg1-Hmg1p in a wild-type (RHY1948), ubc6Δ (RHY2097), and ubc7Δ (RHY2096) genetic background. (E) Fluorescence histogram of strains expressing Deg1-Hmg1p–GFP in a wild-type (RHY1359), hrd1Δ (RHY1566), hrd2-1 (RHY1568), hrd3Δ (RHY1570), and ubc7Δ (RHY1572) genetic background. Strains were analyzed directly from early log-phase cultures. Each histogram represents 10,000 cells. (F) Membrane association of Deg1-Hmg1p. Membrane fractionation of strains expressing Deg1-Hmg1p (RHY1948) is shown. Lysates were spun for 30 min at 4°C to produce the supernatant and pellet fractions. Aliquots of total lysates (T), supernatant fraction (S), and membrane fraction (M) were loaded onto an 8% SDS-PAGE gel, transferred, and immunoblotted with the 9E10 anti-myc antibody.

Figure 4

Vph1p degradation was partially dependent on the HRD genes and UBC7. (A) Degradation of Vph1p in a vma21Δ strain. Results of pulse–chase experiment of VMA21 (RHY566) and vma21Δ (RHY918) strains are shown. Cells were pulse-labeled with ³⁵S-Express for 10 min and chased for the indicated times. Vph1p was immunoprecipitated and analyzed by SDS-PAGE and autoradiography. (B) Pulse–chase experiment of strains containing the vma21Δ allele (parent strain: RHY918) and the hrd1Δ allele (RHY1032), the hrd2-1 allele (RHY1067), the hrd3Δ allele (RHY1034), the ubc6Δ allele (RHY1228), the ubc7Δ allele (RHY1069), the ubc6Δubc7Δ allele (RHY1488), or the hrd1Δ ubc7Δ allele (RHY1491). (C) Densitometric analysis of the pulse–chase experiments in B. Each value is the average of at least two independent pulse–chase experiments. SDs were <10%.

Figure 5

UP* degradation was independent of HRD1 and HRD3 but dependent on UBC7. (A) Cycloheximide–chase assay of strains expressing UP* in a wild-type (RHY1951), hrd1Δ (RHY2094), hrd3Δ (RHY1904), and ubc7Δ (RHY1900) genetic background. After addition of cycloheximide, lysates were prepared at the indicated times and immunoblotted with antiserum generated against the last 10 residues of uracil permease. (B) Levels of UP* ubiquitination correlated with its HRD-independent degradation. Cultures of strains expressing UP* in a wild-type (RHY1216), hrd1Δ (RHY1222), hrd2-1 (RHY1218), hrd3Δ (RHY1223), or ubc7Δ (RHY1221) genetic background were lysed, and UP* was immunoprecipitated with antibodies generated against the N-terminus of uracil permease. Ubiquitination of UP* was assayed by immunoblotting with an anti-ubiquitin antibody.