Grr1 of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skp1: coupling glucose sensing to gene expression and the cell cycle.

Grr1 protein of the yeast Saccharomyces cerevisiae is a central component of a glucose signal transduction mechanism responsible for glucose-induced gene expression. It is required for glucose-stimulated regulation of Rgt1, a repressor of several glucose-induced HXT genes. Grr1 also plays a role in regulating the cell cycle, because it is required for degradation of the G1 cyclins Cln1 and Cln2. We discovered that Grr1 physically interacts with Skp1, a protein that has been implicated in a ubiquitin-conjugating enzyme complex that targets for degradation the cell cycle regulators Cln1 and Cln2, and the cyclin-dependent kinase inhibitor Sic1. Thus, Grr1 may regulate the cell cycle and glucose-induced gene expression via ubiquitin-mediated protein degradation. Consistent with this idea, Skp1, like Grr1, was found to be required for glucose-induced HXT gene expression. Two functional domains of Grr1 are required for its interaction with Skp1: 12 leucine-rich repeats (LRR) and an adjacent F-box. The Grr1-Skp1 interaction is enhanced by high levels of glucose. This could provide yeast with a mechanism for coupling nutrient availability to gene expression and cell cycle regulation.     

Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein

The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region-encoded transport system (reviewed in [1-5]). In addition, A. tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB/VirD4 transport system [6], and both assist there in the transformation of normal cells into tumor cells. The function of the 22 kDa VirF protein is not clear. Deletion of the virF gene in A. tumefaciens leads to diminished virulence [7, 8] and can be complemented by the expression of the virF gene in the host plant. This finding indicates that VirF functions within the plant cell [8]. Here, we report that the VirF protein is the first prokaryotic protein with an F box by which it can interact with plant homologs of the yeast Skp1 protein. The presence of the F box turned out to be essential for the biological function of VirF. F box proteins and Skp1p are both subunits of a class of E3 ubiquitin ligases referred to as SCF complexes. Thus, VirF may be involved in the targeted proteolysis of specific host proteins in early stages of the transformation process.     

Yeast Ran-binding protein Yrb1p is required for efficient proteolysis of cell cycle regulatory proteins Pds1p and Sic1p

Ubiquitin-dependent proteolysis of specific target proteins is required for several important steps during the cell cycle. Degradation of such proteins is strictly cell cycle-regulated and triggered by two large ubiquitin ligases, termed anaphase-promoting complex (APC) and Skp1/Cullin/F-box complex (SCF). Here we show that yeast Ran-binding protein 1 (Yrb1p), a predominantly cytoplasmic protein implicated in nucleocytoplasmic transport, is required for cell cycle regulated protein degradation. Depletion of Yrb1p results in the accumulation of unbudded G(1) cells and of cells arrested in mitosis implying a function of Yrb1p in the G(1)/S transition and in the progression through mitosis. Temperature-sensitive yrb1-51 mutants are defective in APC-mediated degradation of the anaphase inhibitor protein Pds1p and in degradation of the cyclin-dependent kinase inhibitor Sic1p, a target of SCF. Thus, Yrb1p is crucial for efficient APC- and SCF-mediated proteolysis of important cell cycle regulatory proteins. We have identified the UBS1 gene as a multicopy suppressor of yrb1-51 mutants. Ubs1p is a nuclear protein, and its deletion is synthetic lethal with a yrb1-51 mutation. Interestingly, UBS1 was previously identified as a multicopy suppressor of cdc34-2 mutants, which are defective in SCF activity. We suggest that Ubs1p may represent a link between nucleocytoplasmic transport and ubiquitin ligase activity.     

Wheat F-box protein recruits proteins and regulates their abundance during wheat spike development

F-box proteins, components of the Skp1-Cullin1-F-box (SCF) protein E3 ubiquitin ligase complex, serve as the variable component responsible for substrate recognition and recruitment in SCF-mediated proteolysis. F-box proteins interact with Skp1 through the F-box motif and with ubiquitination substrates through C-terminal protein interaction domains. F-box proteins regulate plant development, various hormonal signal transduction processes, circadian rhythm, and cell cycle control. We isolated an F-box protein gene from wheat spikes at the onset of flowering. The Triticum aestivum cyclin F-box domain (TaCFBD) gene showed elevated expression levels during early inflorescence development and under cold stress treatment. TaCFBD green fluorescent protein signals were localized in the cytoplasm and plasma membrane. We used yeast two-hybrid screening to identify proteins that potentially interact with TaCFBD. Fructose bisphosphate aldolase, aspartic protease, VHS, glycine-rich RNA-binding protein, and the 26S proteasome non-ATPase regulatory subunit were positive candidate proteins. The bimolecular fluorescence complementation assay revealed the interaction of TaCFBD with partner proteins in the plasma membranes of tobacco cells. Our results suggest that the TaCFBD protein acts as an adaptor between target substrates and the SCF complex and provides substrate specificity to the SCF of ubiquitin ligase complexes.     

SCF(Skp1-Cul1-F-box蛋白)复合物及其在细胞周期中的作用

泛素 -蛋白酶体降解途径在多种蛋白浓度的调节中发挥重要作用。泛素连接酶家族的一个成员SCF复合物 (Skp1- Cul1- F -box蛋白 )通过降解多个细胞周期调节蛋白促使细胞进入增殖周期。本文综述了SCF复合物的组成和结构以及这一复合物对细胞周期调控因子的调节作用。     

A negatively charged amino acid in Skp2 is required for Skp2-Cks1 interaction and ubiquitination of p27Kip1

Proteolysis of cyclin-dependent kinase inhibitor p27 occurs predominantly in the late G1 phase of the cell cycle through a ubiquitin-mediated protein degradation pathway. Ubiquitination of p27 requires the SCFSkp2 ubiquitin ligase and Skp2 F-box binding protein Cks1. The mechanisms by which Skp2 recognizes Cks1 to ubiquitylate p27 remain obscure. Here we show that Asp-331 in the carboxyl terminus of Skp2 is required for its association with Cks1 and ubiquitination of p27. Mutation of Asp-331 to Ala disrupts the interaction between Skp2 and Cks1. Although Asp-331 mutation negates the ability of the Skp1-Cullin-F-box protein (SCF) complex to ubiquitylate p27, such a mutation has no effect on Skp2 self-ubiquitination. A conservative change from Asp to Glu at position 331 of Skp2 does not affect Skp2-Cks1 interaction. Our results revealed a unique requirement for a negatively charged residue in the carboxyl-terminal region of Skp2 in recognition of Cks1 and ubiquitination of p27.     

A CDK-independent function of mammalian Cks1: targeting of SCF(Skp2) to the CDK inhibitor p27Kip1.

The Cks/Suc1 proteins associate with CDK/cyclin complexes, but their precise function(s) is not well defined. Here we demonstrate that Cks1 directs the ubiquitin-mediated proteolysis of the CDK-bound substrate p27Kip1 by the protein ubiquitin ligase (E3) SCF(Skp2). Cks1 associates with the F box protein Skp2 and is essential for recognition of the p27Kip1 substrate for ubiquitination in vivo and in vitro. Using purified recombinant proteins, we reconstituted p27Kip1 ubiquitination activity and show that it is dependent on Cks1. CKS1-/- mice are abnormally small, and cells derived from them proliferate poorly, particularly under limiting mitogen conditions, possibly due to elevated levels of p27Kip1.     

Structural basis of the Cks1-dependent recognition of p27(Kip1) by the SCF(Skp2) ubiquitin ligase

The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27(Kip1) plays a central role in cell cycle progression, and enhanced degradation of p27(Kip1) is associated with many common cancers. Proteolysis of p27(Kip1) is triggered by Thr187 phosphorylation, which leads to the binding of the SCF(Skp2) (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27(Kip1) ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27(Kip1) phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27(Kip1) binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27(Kip1) is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27(Kip1) to the SCF(Skp2)-Cks1 complex.     

The proteasome/multicatalytic-multifunctional proteinase. In vivo function in the ubiquitin-dependent N-end rule pathway of protein degradation in eukaryotes.

Proteinase yscE, the proteasome/multicatalytic-multifunctional proteinase of yeast had been shown to function in stress response and in the degradation of ubiquitinated proteins [(1991) EMBO J. 10, 555-562]. A well-defined set of proteins degraded via ubiquitin-mediated proteolysis are the substrates of the N-end rule pathway [(1986) Science 234, 179-186; (1989) Science 243, 1576-1583]. We show that mutants defective in the chymotryptic activity of proteinase yscE fail to degrade substrates of the N-end rule pathway. This gives further proof of the proteasome being a central catalyst in ubiquitin-mediated proteolysis.     

A new function of Skp1 in the mitotic exit of budding yeast Saccharomyces cerevisiae

We previously reported that Skp1, a component of the Skp1-Cullin-F-box protein (SCF) complex essential for the timely degradation of cell cycle proteins by ubiquitination, physically interacts with Bfa1, which is a key negative regulator of the mitotic exit network (MEN) in response to diverse checkpoint-activating stresses in budding yeast. In this study, we initially investigated whether the interaction of Skp1 and Bfa1 is involved in the regulation of the Bfa1 protein level during the cell cycle, especially by mediating its degradation. However, the profile of the Bfa1 protein did not change during the cell cycle in skp1-11, which is a SKP1 mutant allele in which the function of Skp1 as a part of SCF is completely impaired, thus indicating that Skp1 does not affect the degradation of Bfa1. On the other hand, we found that the skp1-12 mutant allele, previously reported to block G2-M transition, showed defects in mitotic exit and cytokinesis. The skp1-12 mutant allele also revealed a specific genetic interaction with Deltabfa1. Bfa1 interacted with Skp1 via its 184 C-terminal residues (Bfa1-D8) that are responsible for its function in mitotic exit. In addition, the interaction between Bfa1 and the Skp1-12 mutant protein was stronger than that of Bfa1 and the wild type Skp1. We suggest a novel function of Skp1 in mitotic exit and cytokinesis, independent of its function as a part of the SCF complex. The interaction of Skp1 and Bfa1 may contribute to the function of Skp1 in the mitotic exit.     

The Caenorhabditis elegans Skp1-related gene family: diverse functions in cell proliferation, morphogenesis, and meiosis

BACKGROUND: The SCF ubiquitin-ligase complex targets the ubiquitin-mediated degradation of proteins in multiple dynamic cellular processes. A key SCF component is the Skp1 protein that functions within the complex to link the substrate-recognition subunit to a cullin that in turn binds the ubiquitin-conjugating enzyme. In contrast to yeast and humans, Caenorhabditis elegans contains multiple expressed Skp1-related (skr) genes. RESULTS: The 21 Skp1-related (skr) genes in C. elegans form one phylogenetic clade, suggesting that a single ancestral Skp1 gene underwent independent expansion in C. elegans. The cellular and developmental functions of the 21 C. elegans skr genes were probed by dsRNA-mediated gene inactivation (RNAi). The RNAi phenotypes of the skr genes fall into two classes. First, the highly similar skr-7, -8, -9, and -10 genes are required for posterior body morphogenesis, embryonic and larval development, and cell proliferation. Second, the related skr-1 and -2 genes are required for the restraint of cell proliferation, progression through the pachytene stage of meiosis, and the formation of bivalent chromosomes at diakinesis. CUL-1 was found to interact with SKR-1, -2, -3, -7, -8, and -10 in the yeast two-hybrid system. Interestingly, SKR-3 could interact with both CUL-1 and its close paralog CUL-6. CONCLUSIONS: Members of the expanded skr gene family in C. elegans perform critical functions in regulating cell proliferation, meiosis, and morphogenesis. The finding that multiple SKRs are able to bind cullins suggests an extensive set of combinatorial SCF complexes.     

Degradation of p27(Kip1) at the G(0)-G(1) transition mediated by a Skp2-independent ubiquitination pathway.

Targeting of the cyclin-dependent kinase inhibitor p27(Kip1) for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex. Degradation of p27(Kip1) at the G(0)-G(1) transition of the cell cycle has now been shown to proceed normally in Skp2(-/-) lymphocytes, whereas p27(Kip1) proteolysis during S-G(2) phases is impaired in these Skp2-deficient cells. Degradation of p27(Kip1) at the G(0)-G(1) transition was blocked by lactacystin, a specific proteasome inhibitor, suggesting that it is mediated by the ubiquitin-proteasome pathway. The first cell cycle of stimulated Skp2(-/-) lymphocytes appeared normal, but the second cycle was markedly inhibited, presumably as a result of p27(Kip1) accumulation during S-G(2) phases of the first cell cycle. Polyubiquitination of p27(Kip1) in the nucleus is dependent on Skp2 and phosphorylation of p27(Kip1) on threonine 187. However, polyubiquitination activity was also detected in the cytoplasm of Skp2(-/-) cells, even with a threonine 187 --> alanine mutant of p27(Kip1) as substrate. These results suggest that a polyubiquitination activity in the cytoplasm contributes to the early phase of p27(Kip1) degradation in a Skp2-independent manner, thereby promoting cell cycle progression from G(0) to G(1).