Degradation of the yeast MATα2 transcriptional regulator is mediated by the proteasome

Richter-Ruoff, Birgit; Wolf, Dieter H.; Hochstrasser, Mark

doi:10.1016/0014-5793(94)01085-4

Cited by 52 publications

(33 citation statements)

References 26 publications

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“…Ctk2p turnover is not affected in cells deficient in the major vacuolar peptidases (data not shown), whereas it is markedly affected in a mutant strain deficient for two of the proteasome catalytic subunits (pre1 pre2) (48), indicating that Ctk2p destruction is mediated by the proteasome pathway. Although other cases have been described, targeting to the proteasome usually requires attachment of ubiquitin chains to substrate proteins (47).…”

Section: Discussionmentioning

confidence: 91%

“…Ctk2p turnover was not affected in the vacuolar protease-deficient pra1 prb1 prc1 cps1 mutant strain (17) (data not shown). To establish whether Ctk2p was a substrate for the proteasome degradation pathway, we examined Ctk2p stability in the yeast pre1 pre2 mutant strain, which is defective in two of the proteasome catalytic subunits (48). The CTK2-HA construct was introduced into pre1 pre2 and wild-type isogenic cells.…”

Section: Resultsmentioning

confidence: 99%

“…The yeast strains used in this study were WCG4a (MATa ura3 leu2 -3,112 his3-11,15) and its isogenic pre1-1 pre2-2 derivative WCG4-11/22a, constructed by Richter-Ruoff et al (48), and MHY501 (MAT␣ his3-⌬200 leu2-3,112 ura3-52 lys2-801 trp1-1) and its isogenic ⌬doa4 derivative MHY623, described by Papa and Hochstrasser (45). The ⌬ctk strains were derived from W303-1B (MAT␣ ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura 3-1).…”

Section: Methodsmentioning

confidence: 99%

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The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

Hautbergue

Goguel

1999

Molecular and Cellular Biology

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The yeast CTDK-I complex has been implicated in phosphorylation of the carboxy-terminal domain of the RNA polymerase II and in transcription control. It is composed of three polypeptides: Ctk1p and Ctk2p, a cyclin-dependent kinase and a C-type cyclin subunit, respectively; and Ctk3p, a third subunit of unknown function. Cyclins are regulatory proteins whose expression is tightly controlled at the protein level. In this study, we examined the regulation of Ctk2p expression in vivo. Surprisingly, unlike what has been described for cell cycle cyclins, steady-state levels of Ctk2p are composed of two relatively abundant forms, one of them phosphorylated. We show that this phosphorylated form is extremely unstable (half-life, 5 min) and that rapid proteolysis of Ctk2p exhibits growth-related regulation. Furthermore, our data establish that similar to the case for other naturally short-lived proteins, Ctk2p degradation is mediated by the ubiquitin-proteasome pathway. This is the first demonstration that a C-type cyclin is phosphorylated and targeted to the proteasome. Strikingly, neither phosphorylation nor destruction of Ctk2p requires its associated kinase Ctk1p, a feature fundamentally different from that which has been observed for cell cycle cyclins.Cyclin-dependent protein kinases (CDKs) were first identified as central regulators of the major transitions of the eukaryotic cell division cycle. Their activity is determined by cyclin binding, by both positive and negative regulatory phosphorylation, and by polypeptide CDK inhibitors (40, 41). A single cyclin-dependent kinase subunit associates sequentially with multiple cyclin partners whose abundance fluctuates during the cell cycle (43). Indeed, one aspect of CDK regulation is triggered by the rapid destruction of their cyclin partners (23). Several studies both in yeast and in mammals have demonstrated that cyclins are selectively degraded by the ubiquitindependent proteasome pathway (9,16,53,67). Substrates are first marked for degradation by conjugation with several molecules of ubiquitin, a highly conserved 76-amino-acid-long polypeptide that is joined reversibly by a covalent linkage.

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Section: Discussionmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

Hautbergue

Goguel

1999

Molecular and Cellular Biology

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“…Proteasome inhibition was accomplished through mutations in the PRE1-and PRE2-encoded 20 S subunits; these mutations severely inhibit the proteasome chymotryptic peptidase activity in vitro and retard the degradation of natural and model proteasome substrates in vivo (36,(51)(52)(53)(54). Although the pre1-1 and pre2-2 mutations are maximally inhibitory at 38°C, inhibition of the turnover of many substrates is readily detectable at 30°C (36,52,54).…”

Section: Evidence Against Degradative Signaling By Lys-63 Chains In Vmentioning

confidence: 99%

In Vitro Assembly and Recognition of Lys-63 Polyubiquitin Chains

Hofmann¹,

Pickart²

2001

Journal of Biological Chemistry

234

232

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Polyubiquitin chains assembled through lysine 48 (Lys-48) of ubiquitin act as a signal for substrate proteolysis by 26 S proteasomes, whereas chains assembled through Lys-63 play a mechanistically undefined role in post-replicative DNA repair. We showed previously that the products of the UBC13 and MMS2 genes function in error-free post-replicative DNA repair in the yeast Saccharomyces cerevisiae and form a complex that assembles Lys-63-linked polyubiquitin chains in vitro. Here we confirm that the Mms2⅐Ubc13 complex functions as a high affinity heterodimer in the chain assembly reaction in vitro and report the results of a kinetic characterization of the polyubiquitin chain assembly reaction. To test whether a Lys-63-linked polyubiquitin chain can signal degradation, we conjugated Lys-63-linked tetraubiquitin to a model substrate of 26 S proteasomes. Although the noncanonical chain effectively signaled substrate degradation, the results of new genetic epistasis studies agree with previous genetic data in suggesting that the proteolytic activity of proteasomes is not required for error-free post-replicative repair.

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“…Yeast Strains and Growth Conditions-The yeast strains used in this study were WCG4a (MATa ura3 leu2 -3, 112 his3-11, 15) and its isogenic pre1-1 pre2-2 derivative WCG4-11/22a constructed by Richter-Ruoff et al (55). The ⌬ctk strains (MAT␣ ade2-1 can1-100 his3 -11, 15 leu2-3, 112 trp1-1 ura 3-1, ctk::TRP1) are described in work by Hautbergue and Goguel (45).…”

Section: Methodsmentioning

confidence: 99%

Activation of the Cyclin-dependent Kinase CTDK-I Requires the Heterodimerization of Two Unstable Subunits

Hautbergue

Goguel

2001

Journal of Biological Chemistry

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RNA polymerase II CTD kinases are key elements in the control of mRNA synthesis. They constitute a family of cyclin-dependent kinases activated by C-type cyclins. Unlike most cyclin-dependent kinase complexes, which are composed of a catalytic and a regulatory subunit, the yeast CTD kinase I complex contains three specific subunits: a kinase subunit (Ctk1), a cyclin subunit (Ctk2), and a third subunit (Ctk3) of unknown function that does not exhibit any similarity to known proteins. Like the Ctk2 cyclin that is regulated at the level of protein turnover, Ctk3 is an unstable protein processed through a ubiquitin-proteasome pathway. Interestingly, Ctk2 and Ctk3 physical interaction is required to protect both subunits from degradation, pointing to a new mechanism for cyclin turnover regulation. We also show that Ctk2 and Ctk3 can each interact independently with the kinase. However, despite the formation of CDK/ cyclin complexes in vitro, the Ctk2 cyclin is unable to activate its CDK: both Ctk2 and Ctk3 are required for Ctk1 CTD kinase activation. The different specific features governing CTDK-I regulation probably reflect requirement for the transcriptional response to multiple growth conditions. The C-terminal domain (CTD) 1 of the large subunit of RNA polymerase II contains a repeated heptapeptide that is highly phosphorylated in a portion of the molecules in the cell. This domain plays an essential role in the control of mRNA synthesis, as well as RNA processing, and its phosphorylation is a key feature of its function (1). The formation of initiation complexes on promoter DNA involves RNA polymerase II molecules with unphosphorylated CTD, whereas the CTD becomes phosphorylated during or after the transition to elongation. Three CTD kinases are involved in the regulation of RNA polymerase II transcription in the yeast Saccharomyces cerevisiae, all being cyclin-dependent kinases (CDKs).The first yeast CTD kinase, Kin28 (2), is associated with cyclin Ccl1 (3) and is required for transcription of most genes (4). This complex, like its human counterpart the Cdk7 kinase complex (5), is a component of the general transcription factor TFIIH (6, 7). Kin28 is an essential kinase that has been shown to phosphorylate the CTD only after the RNA polymerase II is associated with promoter DNA, thus promoting the elongation mode (8). Kin28 is also required for proper capping enzyme targeting in vivo (9). The second yeast CDK implicated in CTD phosphorylation, the nonessential Srb10/Ume5/Ssn3 kinase and its cyclin partner Srb11/Ume3/Ssn8, is a component of the holoenzyme (10). The Srb10/11 complex is structurally related to mammalian Cdk8 kinase and its associated cyclin C (11). Several studies established that the Srb10 kinase is implicated in the transcriptional repression of specific sets of genes (4, 12-15). Hengartner et al. (8) showed that Srb10 is uniquely capable of phosphorylating the CTD prior to formation of the initiation complex on promoter DNA, with consequent inhibition of transcription. Srb10 has also been i...

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Degradation of the yeast MATα2 transcriptional regulator is mediated by the proteasome

Cited by 52 publications

References 26 publications

The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

In Vitro Assembly and Recognition of Lys-63 Polyubiquitin Chains

Activation of the Cyclin-dependent Kinase CTDK-I Requires the Heterodimerization of Two Unstable Subunits

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