Degradation of Ornithine Decarboxylase by the Mammalian and Yeast 26S Proteasome Complexes Requires all the Components of the Protease

Elias, Sarah; Bercovich, Beatrice; Kahana, Chaim; Coffino, Philip; Fischer, Michael B.; Hilt, Wolfgang; Wolf, Dieter H.; Ciechanover, Aaron

doi:10.1111/j.1432-1033.1995.tb20466.x

Cited by 13 publications

(16 citation statements)

References 29 publications

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“…Together, these results suggest that proteolysis of p␣F by the proteasome complex may take place by a ubiquitinindependent pathway, as observed for ornithine decarboxylase (39,40) and casein (41). It is also known that full-length proteins can be fully degraded to peptides in the absence of ubiquitin by the 20S proteasome if the proteins are first denatured (ref.…”

Section: Erad Is Not Inhibited By Mutations In Specific Ubiquitinmentioning

confidence: 83%

Proteasome-dependent endoplasmic reticulum-associated protein degradation: An unconventional route to a familiar fate

Werner

Brodsky

McCracken

1996

Proc. Natl. Acad. Sci. U.S.A.

424

314

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Until recently, the degradation of aberrant and unassembled proteins retained in the endoplasmic reticulum (ER) was thought to involve unidentified ER-localized proteases. We now show that the ER-associated degradation (ERAD) of two mutant proteins that accumulate in the ER lumen is inhibited in a proteasome-defective yeast strain and when cytosol from this mutant is used in an in vitro assay. In addition, ERAD is limited in vitro in the presence of the proteasome inhibitors, 3,4-dichloroisocoumarin and lactacystin. Furthermore, we find that an ERAD substrate is exported from ER-derived microsomes, and the accumulation of exported substrate is 2-fold greater when proteasome mutant cytosol is used in place of wild-type cytosol. We conclude that lumenal ERAD substrates are exported from the yeast ER to the cytoplasm for degradation by the proteasome complex.Quality control of newly synthesized proteins in the endoplasmic reticulum (ER) ensures that only correctly folded, processed, and completely assembled proteins exit this compartment for further transport through the secretory pathway. Most proteins that fail to reach this transport competent state are degraded (1-7). The ER-associated protein degradation pathway (ERAD) is highly selective for specific unassembled and͞or aberrant proteins, while the majority of ER resident and secreted proteins are quite stable. Evidence that the ER chaperone calnexin has a role in ERAD indicates that molecular chaperones might be required for this remarkable substrate selectivity (8).Although previous studies suggested that ERAD involves unidentified proteases localized in the ER, more recent evidence indicates that ERAD may require cytosolic proteases. Studies of ERAD in vitro suggest that degradation of aberrant ER-lumenal proteins occurs in the cytoplasm (8), and there is also evidence that the degradation of ER-retained forms of the cystic fibrosis transmembrane conductance regulator is ubiquitin-and proteasome-dependent (9, 10). In addition, cytomegalovirus-induced down-regulation of major histocompatibility complex class I molecules involves the rapid transport of the unassembled major histocompatibility complex I heavy chains from the ER to the cytoplasm for degradation by the proteasome complex (11). In this report we show that the cytosolic proteasome complex is the proteolytic component for the ER-associated degradation of two ER-lumenal proteins in yeast. Furthermore, we provide evidence that indicates ERAD substrates are exported to the cytosol for degradation by the proteasome. MATERIALS AND METHODSMaterials and Strains. Strains used were: RSY607 (Mat␣, pep4::URA3), provided by R. Schekman, University of California, Berkeley; AB122 (Mata, prc1-407, prb1-1122, pep4-3, leu2, ura3-52), provided by A. Brake, University of California, San Francisco; pre 1-1 pre 2-2 proteasome mutant and isogenic wild-type strain, provided by D. H. Wolf (12, 13); ⌬ubc6 ubiquitin-conjugating enzyme mutant and isogenic wild-type strains, provided by S. Jentsch (14); and ubc4-⌬1 u...

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Section: Erad Is Not Inhibited By Mutations In Specific Ubiquitinmentioning

confidence: 83%

Proteasome-dependent endoplasmic reticulum-associated protein degradation: An unconventional route to a familiar fate

Werner

Brodsky

McCracken

1996

Proc. Natl. Acad. Sci. U.S.A.

424

314

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“…However, this result alone does not rule out Sp1 ubiquitination, in that Sp1 is a relatively large protein and a small amount of ubiquitin would not significantly alter the mobility of Sp1 in SDS-PAGE. Other proteins (c-Jun [26] and ornithine decarboxylase [18]) have been shown to be subject to proteasome degradation in the absence of ubiquitination.…”

Section: Resultsmentioning

confidence: 99%

Reduced O Glycosylation of Sp1 Is Associated with Increased Proteasome Susceptibility

Han

Kudlow

1997

Molecular and Cellular Biology

401

344

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Sp1 is a ubiquitously expressed transcription factor that is particularly important for the regulation of TATA-less genes that encode housekeeping proteins. Most growth factors and receptors are also encoded by such genes. Sp1 is multiply O glycosylated by covalent linkage of the monosaccharide N-acetylglucosamine (O-GlcNAc) to serine and threonine residues. Based on an earlier observation that growth factor gene transcription can be regulated by glucose and glucosamine in vascular smooth muscle cells, we determined whether Sp1 glycosylation could be regulated and if this modification altered Sp1 function. We found that Sp1 becomes hyperglycosylated when cells are exposed to 5 mM glucosamine, whereas under glucose starvation, stimulation with cyclic AMP (cAMP) results in nearly complete deglycosylation of this protein. Correlating with this hypoglycosylated state, Sp1 is rapidly proteolytically degraded by an enzyme(s) that can be inhibited by specific proteasome inhibitors, lactacystin and LLnL. Treatment of cells with glucose or glucosamine protects Sp1 from cAMP-mediated degradation, whereas blockade of glucosamine synthesis abrogates glucose but not glucosamine protection. This effect on Sp1 is specific, in that the Stat-3 and E2F transcription factors did not undergo degradation under these conditions. The O-GlcNAc modification of Sp1 may play a role as a nutritional checkpoint. In the absence of adequate nutrition, Sp1 becomes hypoglycosylated and thereby subject to proteasome degradation. This process could potentially result in reduced general transcription, thereby conserving nutrients.

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“…The turnover of ODC, which is mediated by a polyamine-inducible protein termed antizyme, has been studied extensively (25)(26)(27)(28)(29), but there is no published information available on the mechanism of degradation of S-adenosylmethionine decarboxylase and SSAT.…”

mentioning

confidence: 99%

Proteasomal Degradation of Spermidine/Spermine N 1-Acetyltransferase Requires the Carboxyl-terminal Glutamic Acid Residues

Coleman¹,

Pegg

1997

Journal of Biological Chemistry

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The rapid turnover of spermidine/spermine N 1 -acetyltransferase (SSAT), a key enzyme in the regulation of polyamine levels, was found to be mediated via ubiquitination and the proteasomal system. SSAT degradation was blocked by the binding of polyamines or of the polyamine analog, N 1 ,N 12 -bis(ethyl)spermine (BE-3-4-3), to the protein, providing a mechanism for the increase of SSAT activity in response to these agents. Site-directed mutagenesis indicated that a number of residues including arginine 19, cysteine 122, histidine 126, glutamic acid 152, arginine 155, and methionine 167 were needed for protection of SSAT by BE-3-4-3. These residues have previously been shown to reduce the affinity for the binding of polyamines to the SSAT protein, and these results indicate that the change in protein configuration brought about by this binding renders the protein resistant to proteasomal degradation. Mutations to alanines of residues arginine 7, cysteine 14, and lysine 141 also prevented the protection by BE-3-4-3, and these residues may be required for the formation of the protected conformation. The rapid degradation of SSAT required the carboxyl-terminal region of the protein, and the two terminal glutamic acid residues at positions 170 and 171 were found to be of critical importance. Truncation of the protein to remove these residues or the mutation of either of these acidic residues to glutamine completely abolished the rapid degradation of SSAT. The addition of two extra lysine residues at the carboxyl terminus or the conversion of the glutamic acids at positions 170 and 171 to lysines also prevented SSAT degradation by the proteasome. These results show the key role of the acidic residues at the carboxyl terminus of the protein in reacting with the proteasome. In contrast, mutation of lysine 166 to alanine, which extends the length of the acidic region in the carboxyl-terminal fragment of SSAT, actually increased the rate of degradation of SSAT without affecting its stabilization by BE-3-4-3. The binding of BE-3-4-3 or polyamines is therefore likely to change the configuration of the SSAT protein in a way that prevents the exposure of the carboxyl-terminal region of the ubiquitinated protein to the proteasome.Spermidine/spermine N 1 -acetyltransferase (SSAT), 1 which converts spermidine and spermine into their N 1 -acetyl derivatives, is an important enzyme in mammalian cells that prevents the overaccumulation of polyamines by facilitating their excretion and degradation (1-3). Alterations in SSAT activity are brought about by changes in amount of enzyme protein (4, 5), and the enzyme is highly inducible by a variety of hormones, physiological stimuli, drugs, and toxic agents. It is also strongly induced by polyamines, and it has been suggested that a rise in the free polyamine content is an intermediary in the induction by other agents (6). The most potent inducers of SSAT are polyamine analogs that have substitutions on the terminal nitrogens and, therefore, are not substrates for acetylation (1,(7)(8)(9)...

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Degradation of Ornithine Decarboxylase by the Mammalian and Yeast 26S Proteasome Complexes Requires all the Components of the Protease

Cited by 13 publications

References 29 publications

Proteasome-dependent endoplasmic reticulum-associated protein degradation: An unconventional route to a familiar fate

Proteasome-dependent endoplasmic reticulum-associated protein degradation: An unconventional route to a familiar fate

Reduced O Glycosylation of Sp1 Is Associated with Increased Proteasome Susceptibility

Proteasomal Degradation of Spermidine/Spermine N 1-Acetyltransferase Requires the Carboxyl-terminal Glutamic Acid Residues

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