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. 2000 Jul;20(13):4691-8.
doi: 10.1128/mcb.20.13.4691-4698.2000.

Evidence for an Interaction Between Ubiquitin-Conjugating Enzymes and the 26S Proteasome

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Free PMC article

Evidence for an Interaction Between Ubiquitin-Conjugating Enzymes and the 26S Proteasome

P Tongaonkar et al. Mol Cell Biol. .
Free PMC article

Abstract

The targeting of proteolytic substrates is accomplished by a family of ubiquitin-conjugating (E2) enzymes and a diverse set of substrate recognition (E3) factors. The ligation of a multiubiquitin chain to a substrate can promote its degradation by the proteasome. However, the mechanism that facilitates the translocation of a substrate to the proteasome in vivo is poorly understood. We have discovered that E2 proteins, including Ubc1, Ubc2, Ubc4, and Ubc5, can interact with the 26S proteasome. Significantly, the interaction between Ubc4 and the proteasome is strongly induced by heat stress, consistent with the requirement for this E2 for efficient stress tolerance. A catalytically inactive derivative of Ubc4 (Ubc4(C86A)), which causes toxicity in yeast cells, can also bind the proteasome. Purified proteasomes can ligate ubiquitin to a test substrate without the addition of exogenous E2 protein, suggesting that the ubiquitylation of some proteolytic substrates might be directly coupled to degradation by the proteasome.

Figures

FIG. 1
FIG. 1
Ubc4 interacts with the proteasome. (A) Protein extracts were prepared from JD47-13C that expressed Pre1-FLAG and separated on a Sepharose-4B column. The fractions were examined by immunoblotting using antibodies against Ubc4 and FLAG. The fraction numbers and positions of gel filtration standards (in kilodaltons) are shown. Fractions 14 to 18 correspond to the column void volume. (B) An equal volume from the indicated Sepharose-4B fractions was incubated with FLAG-agarose, and the precipitated proteins were examined in an immunoblot with antibodies against Ubc4. (C) Protein extracts were separated on a Superose-6 column, and the migration of Pre1-FLAG and Ubc4 was determined as in panel A. (D) The Ubc4-proteasome interaction was examined by immunoprecipitating the proteasome from a wild-type strain (PHY209). Immunoprecipitation of 2μm plasmid-based Pre1-FLAG yielded high levels of Ubc4 (lane 1, Immunoprecipitate). However, the recovery of Pre1-FLAG-6His (lane 2), Pre2-HA (lane 3), and Pup1-HA (lane 4) expressed at single-copy levels yielded much lower levels of the proteasome as well as Ubc4, due to inefficient antibody reactions. Ubc4 was not precipitated from a control extract that did not express an epitope-tagged protein (lane 5). The expression of Ubc4 was similar in all the strains (Extract).
FIG. 2
FIG. 2
Ubc4 and subunits in the 19S and 20S particles can be immunoprecipitated with Pre1-FLAG. (A) The proteasome subunits Rpn8-V5 (19S) and Pup2-V5 (20S) could be precipitated on FLAG-agarose beads, and detected with V5 antibodies, from extracts that expressed Pre1-FLAG (lanes 2 and 4, respectively). Despite equivalent levels of expression (lanes 5 to 8), neither Pup2-V5 nor Rpn8-V5 was precipitated from extracts that lacked Pre1-FLAG (lanes 1 and 3). (B) Ubc4 was detected in extracts prepared from wild-type (lanes 1 and 2) and ubc5Δ strains (lane 4) but not in ubc4Δ (lane 3), or ubc4Δ ubc5Δ strains (lane 5). Ubc4 was coimmunoprecipitated only from strains that contained Pre1-FLAG (lanes 2 and 4, FLAG-IP) and not from a strain that did not express Pre1-FLAG (lane 1, FLAG-IP). The same immunoblot was incubated with antibodies against Rpt1 and Ubc2, and, as expected, both proteins were recovered only from the strains that expressed Pre1-FLAG (lanes 2 to 5). (C) Multiple E2 enzymes associate with the proteasome. Extracts prepared from a wild-type yeast strain containing (+) or lacking (−) Pre1-FLAG were separated by SDS-PAGE, and a single immunoblot was incubated sequentially with antibodies against Rpt1, Ubc4, Ubc1, and Ubc2. The expression of Pre1-FLAG did not alter the level of Rpt1 or the E2 proteins (lane 1 and 2, Extract). Equal amounts of extract were incubated with FLAG-agarose, and Rpt1, Ubc4, Ubc1, and Ubc2 were precipitated from the strain that expressed Pre1-FLAG (lane 4, α-FLAG). (The asterisk indicates a cross-reaction against the immunoglobulin light chain, which has a higher mobility than Ubc1.) (D) Samples were prepared as in panel C, and the beads were incubated for 15 min at 30°C with buffer containing (+) or lacking (−) FLAG peptide. Proteins present in the supernatant and beads were separated by SDS-PAGE and examined by immunoblotting. Pre1-FLAG, Rpt1, and Ubc4 were quantitatively released following exposure to FLAG peptide (lane 2). In contrast, treatment with buffer alone resulted in the retention of all three proteins on the FLAG-agarose beads (lane 3).
FIG. 3
FIG. 3
High-copy expression and heat stress result in an increased E2-proteasome interaction. (A) UBC4 and UBC2 were expressed from the galactose-inducible GAL1 promoter in wild-type yeast (JD47-13C). With the exception of lanes 1 and 5, all the strains also expressed Pre1-FLAG (PHY209). The effect of overexpressing Ubc4 was examined in lanes 3 and 7, while the effect of overexpressing Ubc2 was tested in lanes 4 and 8. The expression of the endogenous genes was observed in extracts prepared from cells grown in glucose medium (Glucose panel, lanes 1 to 4). Equal amounts of extract were incubated with FLAG-agarose, and the precipitated proteins were analyzed by immunoblotting (FLAG-IP). Ubc4 was recovered from extracts that contained Pre1-FLAG (lanes 2 to 4) but not from a strain that did not express this epitope-tagged proteasome subunit (lane 1). A similar analysis of extracts prepared from galactose-grown cultures showed that high-level expression of Ubc4 (lane 7, Extract) resulted in an increased proteasome interaction (lane 7, FLAG-IP). Interestingly, overexpression of Ubc2 (lane 8, Extract) did not affect the Ubc4-proteasome interaction noticeably (lane 8, FLAG-IP). Overexpression of Ubc2 also led to its increased interaction with the proteasome (see Fig. 4A). (B) We also examined the effect of PGAL1::UBC1 on the Ubc4-proteasome interaction. In agreement with the results in panel A, growth of PHY212 in galactose medium led to higher expression of Ubc1 (lane 2) and an increased proteasome interaction (lane 6). However, the expression of endogenous Ubc4 (lanes 3 and 4) and its interaction with the proteasome (lanes 7 and 8) were unaffected. Even-numbered lanes represent extracts prepared from PHY212 that expressed PGAL1::UBC1, while the extracts examined in the odd-numbered lanes represent a control that contained vector. Note that endogenous Ubc1 was also detected in the proteasome (lane 5). (C) Heat stress increased the Ubc4-proteasome interaction. Yeast cells expressing Pre1-FLAG were grown at 23°C for 24 h and then incubated at 23, 30, and 37°C for 2 h. We found that the cellular levels of Ubc4 increased ∼twofold at 37°C (Extract). However, the level of Ubc4 that was bound to the proteasome increased ∼25-fold when the temperature was increased from 23 to 37°C. The relative increase in the levels of Ubc4 in the proteasome was >10-fold. In contrast, the recovery of Rpt1 with Pre1-FLAG was unaffected at the different temperatures. (D) A catalytically inactive derivative of Ubc4 (Ubc4C86A) can interact with the proteasome. Extracts prepared from the ubc4Δ ubc5Δ mutant that expressed both Pre1-FLAG and Ubc4C86A (lane 3, Extract) were incubated with FLAG-agarose. Ubc4C86A was precipitated with the proteasome (lane 3, FLAG-IP), although its intracellular levels were much lower than those of wild-type (WT) Ubc4 (lane 2). Lane 1 represents a control strain that did not express Pre1-FLAG.
FIG. 4
FIG. 4
Ub-conjugating activity is detected in immunopurified proteasomes. (A) Protein extracts were prepared from galactose-grown cells that contained PGAL1::UBC2 (lanes 3, 4, 7, and 8) or a vector (lanes 1, 2, 5, and 6). The samples examined in the even-numbered lanes also contained Pre1-FLAG. Equal amounts of extract were incubated with FLAG-agarose, and the level of Ubc2 in the proteasome was determined with specific antibodies. Ubc2 was detected in the proteasome only in the strains that expressed Pre1-FLAG (lanes 6 and 8), although only the sample that contained overexpressed Ubc2 is visible in this reproduction (lane 8). (B) 32P-Ub was prepared as described previously (32), and reaction mixtures containing purified E1, 32P-Ub, and histone H2B were incubated with immunopurified proteasomes. The ubiquitylation reactions were terminated by the addition of SDS-containing gel-loading buffer, and the products were separated in an SDS-polyacrylamide gel and exposed to X-ray film. Lane 1 contains only 32P-Ub, and the contents of the various reaction mixtures are indicated. Significant ubiquitylation of H2B was detected in extracts that contained high levels of Ubc2 precipitated with Pre1-FLAG (lane 9). (C) We estimated the amount of Ubc2 that could be recovered with Pre1-FLAG from PHY211. Protein extracts (3 mg) were prepared from galactose-grown PHY211 and incubated with FLAG-agarose. The beads were washed, suspended in SDS sample buffer, and resolved by SDS-PAGE. We also separated various amounts of recombinant Ubc2 in the same gel (lanes 3 to 5). The resolved proteins were transferred to nitrocellulose and examined with antibodies against Ubc2. We recovered ∼10 ng of Ubc2 with Pre1-FLAG (lane 1). A low level of Ubc2 could also be detected in extracts prepared from noninduced cells (lane 2). (D) We examined the ubiquitylation of H2B by recombinant Ubc2 and proteasome-associated Ubc2. We prepared a standard reaction mixture that contained recombinant Ubc2 (250 ng), E1, 32P-Ub and histone H2B (lane 1). A similar reaction mixture containing proteasome-associated Ubc2 was prepared from ∼25 mg of cell extract that was derived from galactose-grown PHY211 (lane 2). An equivalent reaction mixture (containing proteasome-associated Ubc2) was supplemented with excess unlabeled Ub (lane 3). Proteasome-associated Ubc2 appeared to generate more high-molecular-mass 32P-Ub–H2B conjugates (lane 2) than did recombinant Ubc2 (lane 1). Significantly, the generation of multiply 32P-ubiquitylated H2B by proteasome-associated Ubc2 was strongly inhibited in the presence of excess unlabeled Ub.

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