New insights into cyclins, CDKs, and cell cycle control

Since their initial discovery in yeast, cyclin-dependent kinases have proven to be universal regulators of the cell cycle in all eukaryotes. In unicellular eukaryotes, cell cycle progression is principally governed by one catalytic subunit (cyclin-dependent kinase) that pairs with cell cycle-specific regulatory subunits known as cyclins. Progression through a specific phase of the cell cycle is under the control of a specific class of cyclin. Cell cycle control in multicellular eukaryotes has an additional layer of complexity, as multiple CDKs and cyclins are required. In this review, we will discuss recent advances in the area of cyclins and CDKs, with emphasis on the role of the mammalian proteins in cell cycle control at the cellular and at the organismal level. Many recent surprises have come to light recently as a result of genetic manipulation of cells and mice, and these findings suggest that our understanding of the intricacies of the cell cycle is still rudimentary at best.     

Novel protein phosphatases in yeast.

During the last decade several novel yeast genes encoding proteins related to the PPP family of Ser/Thr protein phosphatases have been discovered and their functional characterization initiated. Most of these novel phosphatases display intriguing structural features and/or are involved in a number of important functions, such as cell cycle regulation, protein synthesis and maintenance of cellular integrity. While in some cases these genes appear to be restricted to fungi, in others similar proteins can be found in higher eukaryotes. This review will summarize the latest advances in our understanding about how these phosphatases are regulated and fulfil their functions in the yeast cell.     

Evolution of networks and sequences in eukaryotic cell cycle control

The molecular networks regulating the G1-S transition in budding yeast and mammals are strikingly similar in network structure. However, many of the individual proteins performing similar network roles appear to have unrelated amino acid sequences, suggesting either extremely rapid sequence evolution, or true polyphyly of proteins carrying out identical network roles. A yeast/mammal comparison suggests that network topology, and its associated dynamic properties, rather than regulatory proteins themselves may be the most important elements conserved through evolution. However, recent deep phylogenetic studies show that fungal and animal lineages are relatively closely related in the opisthokont branch of eukaryotes. The presence in plants of cell cycle regulators such as Rb, E2F and cyclins A and D, that appear lost in yeast, suggests cell cycle control in the last common ancestor of the eukaryotes was implemented with this set of regulatory proteins. Forward genetics in non-opisthokonts, such as plants or their green algal relatives, will provide direct information on cell cycle control in these organisms, and may elucidate the potentially more complex cell cycle control network of the last common eukaryotic ancestor.     

Pleiotropic functions of the yeast Greatwall-family protein kinase Rim15p: a novel target for the control of alcoholic fermentation

Rim15p, a Greatwall-family protein kinase in yeast Saccharomyces cerevisiae, is required for cellular nutrient responses, such as the entry into quiescence and the induction of meiosis and sporulation. In higher eukaryotes, the orthologous gene products are commonly involved in the cell cycle G₂/M transition. How are these pleiotropic functions generated from a single family of protein kinases? Recent advances in both research fields have identified the conserved Greatwall-mediated signaling pathway and a variety of downstream target molecules. In addition, our studies of S. cerevisiae sake yeast strains revealed that Rim15p also plays a significant role in the control of alcoholic fermentation. Despite an extensive history of research on glycolysis and alcoholic fermentation, there has been no critical clue to artificial modification of fermentation performance of yeast cells. Our finding of an in vivo metabolic regulatory mechanism is expected to provide a major breakthrough in yeast breeding technologies for fermentation applications.     

Inhibition of protein synthesis by TOR inactivation revealed a conserved regulatory mechanism of the BiP chaperone in Chlamydomonas

The target of rapamycin (TOR) kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates with highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here, we report that down-regulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis, since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP, such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function, as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification.     

酵母TOR信号转导途径

TOR(target of rapamycin)是真核细胞中一种高度保守的与磷脂酰肌醇激酶相关的蛋白激酶(PIKK),它是免疫抑制剂/抗癌药物雷帕霉素(rapamycin)的靶物质。TOR是细胞生长的中枢控制因子,外界营养因素通过TOR的作用控制酵母、果蝇和哺乳动物细胞的生长。TOR根据细胞环境的营养条件做出相应的应答,参与调控蛋白激酶和蛋白磷酸酯酶的活性,从而控制与蛋白质合成和基因转录相关基因的表达。现对酵母细胞中TOR信号转导途径的研究进行简明的阐述。     

cdc2-related protein kinases and cell cycle control in trypanosomatids

The molecular mechanisms that control the cell cycle have been studied extensively in yeast and higher eukaryotes. Investigations have centred on the cyclin-dependent kinase family of serine/threonine protein kinases, the best characterized of which is cdc2, a key regulatory element in the control of mitosis. Cell cycle control plays an important role in trypanosomes and Leishmania, not only in cellular proliferation, but also in the developmental system that controls the transfer of the parasite between hosts. In this review, Jeremy Mottram compares the family of trypanosome cdc2-related kinases with that of yeast and the higher eukaryotes.     

A ubiquitin conjugating enzyme encoded by African swine fever virus.

The post-translational modification of proteins by covalent attachment of ubiquitin occurs in all eukaryotes by a multi-step process. A family of E2 or ubiquitin conjugating (UBC) enzymes catalyse one step of this process and these have been implicated in several diverse regulatory functions. We report here the sequence of a gene encoded by African swine fever virus (ASFV) which has high homology with UBC enzymes. This ASFV encoded enzyme has UBC activity when expressed in Escherichia coli since it forms thiolester bonds with [125I]ubiquitin in the presence of purified ubiquitin activating enzyme (E1) and ATP, and subsequently transfers [125I]ubiquitin to specific protein substrates. These substrates include histones, ubiquitin and the UBC enzyme itself. The ASFV encoded UBC enzyme is similar in structure and enzyme activity to the yeast ubiquitin conjugating enzymes UBC2 and UBC3. This is the first report of a virus encoding a functionally active UBC enzyme and provides an example of the exploitation of host regulatory mechanisms by viruses.     

Cellular response to unfolded proteins in the endoplasmic reticulum of plants

Secretory and transmembrane proteins are synthesized in the endoplasmic reticulum (ER) in eukaryotic cells. Nascent polypeptide chains, which are translated on the rough ER, are translocated to the ER lumen and folded into their native conformation. When protein folding is inhibited because of mutations or unbalanced ratios of subunits of hetero-oligomeric proteins, unfolded or misfolded proteins accumulate in the ER in an event called ER stress. As ER stress often disturbs normal cellular functions, signal-transduction pathways are activated in an attempt to maintain the homeostasis of the ER. These pathways are collectively referred to as the unfolded protein response (UPR). There have been great advances in our understanding of the molecular mechanisms underlying the UPR in yeast and mammals over the past two decades. In plants, a UPR analogous to those in yeast and mammals has been recognized and has recently attracted considerable attention. This review will summarize recent advances in the plant UPR and highlight the remaining questions that have yet to be addressed.     

Complexity of Mitotic Exit

Completion of mitosis in budding yeast is triggered by activation of the protein phosphatase Cdc14, which is the ultimate effector of a signalling cascade, known as the mitotic exit network. Cdc14 activation leads to eradication of mitotic kinase activity, which is pivotal for mitotic exit and cytokinesis in all eukaryotes. The complexity in mitotic exit regulation is underscored by the recent discovery of a novel network, the so-called FEAR pathway that regulates early Cdc14 activation. Surprisingly, this has revealed an unexpected role for Spo12, a protein involved in meiosis, in Cdc14 activation. In this review, we will discuss these findings together with recent advances in deciphering the function of the FEAR circuit, which has unravelled an exciting new side of Cdc14.

Key Words:

Mitotic exit, Cdc14 activation, FEAR pathway, Spo12, Budding yeast     

Actin-binding proteins: how to reveal the conformational changes

Actin is the most abundant protein in eukaryotes. Under physiological conditions, it can polymerize into polarized filaments. At the heart of these processes are actin-binding proteins that stimulate actin assembly. Most of them are composed of multiple domains that perform both regulatory and signaling functions. Many actin-binding proteins, including WASP and formin family proteins, are auto-inhibited through intramolecular interactions that mask the actin-regulating sites of these proteins. The large flexible molecules of formins have so far eluded crystallization, and have been crystallized only partially. The information from the available crystal structures is valuable, but somewhat difficult to interpret without a larger framework on which to pose the actin-binding mechanism. Single-particle electron microscopy and electron tomography could provide such a large framework with the full-length structures of protein complexes. The recent advances in determining the molecular interactions in protein complexes predict that the molecular modeling and molecular dynamics methods could be employed to study conformational changes in molecules.     

The Mechanism of Eukaryotic Translation Initiation: New Insights and Challenges

Translation initiation in eukaryotes is a highly regulated and complex stage of gene expression. It requires the action of at least 12 initiation factors, many of which are known to be the targets of regulatory pathways. Here we review our current understanding of the molecular mechanics of eukaryotic translation initiation, focusing on recent breakthroughs from in vitro and in vivo studies. We also identify important unanswered questions that will require new ideas and techniques to solve.This work aims to present the current state of our knowledge of the molecular mechanics of translation initiation in eukaryotes. We focus on advances that have taken place over the last few years and, because of space limitations, assume readers will be able to find references to the foundational literature for the field (published before 2000) in the more recent works that are cited here. As always, we apologize for not having the space to cite many important works. Please view this as merely an introduction to the field rather than a complete summary.     

The molecular era of protein S-acylation: spotlight on structure,mechanisms, and dynamics

S-Acylation (commonly referred to as S-palmitoylation) is a post-translational modification consisting in the covalent attachment of an acyl chain to a cysteine residue of the target protein. The lability of the resulting thioester bond gives S-acylation an essential characteristic: its reversibility. S-acylation dynamically regulates different aspects in the life of a protein (including stability, localization, interactome, and function) and, thus, plays critical roles in cellular physiology. For long, the reversibility of S-acylation has been neglected and thereby its potential as a regulatory mechanism for protein function undervalued. Thanks to technological advances, the field has now entered its golden era. A great diversity of interesting targets is being identified, the physio-pathological importance of the modification is starting to be revealed, structural information on the enzymes is becoming available, and the regulatory dynamics are gradually being understood. Here we will review the most recent literature in the S-acylation field, with a special focus on the molecular aspects of the modification, its regulation, and its consequences.     

Complexity of mitotic exit

Completion of mitosis in budding yeast is triggered by activation of the protein phosphatase Cdc14, which is the ultimate effector of a signalling cascade, known as the mitotic exit network. Cdc14 activation leads to eradication of mitotic kinase activity, which is pivotal for mitotic exit and cytokinesis in all eukaryotes. The complexity in mitotic exit regulation is underscored by the recent discovery of a novel network, the so-called FEAR pathway that regulates early Cdc14 activation. Surprisingly, this has revealed an unexpected role for Spo12, a protein involved in meiosis, in Cdc14 activation. In this review, we will discuss these findings together with recent advances in deciphering the function of the FEAR circuit, which has unravelled an exciting new side of Cdc14.     

Yeast ferrochelatase: expression in a baculovirus system and purification of the expression protein.

The terminal step of the heme biosynthetic pathway is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). In eukaryotes this enzyme is bound to the inner mitochondrial membrane with its active site facing the matrix side of the membrane. Previously this laboratory has characterized this enzyme via kinetic and protein chemical modification techniques, and with the recent cloning of the enzyme from yeast, mouse, and human sources it now becomes possible to approach structure-function questions by using site-directed mutagenesis. Of primary significance to this is the development of an efficient expression vector. This is of particular significance for ferrochelatase, as it is a low-abundance protein whose DNA coding sequence has a very low codon bias. In the current work we describe the production of yeast ferrochelatase in a baculovirus system. This system is shown to be an excellent one in which to produce large quantities of active ferrochelatase. The expressed enzyme is membrane associated and is not released into the growth medium either during or after virus development and cell lysis. The expressed protein can be purified in a procedure that requires only 1 day and makes use of a Pharmacia Hi Trap blue affinity column. The measured Km's for the substrates mesoporphyrin and iron are the same as those reported previously for the yeast enzyme. To our knowledge this is the first example of a mitochondrial membrane protein that has been expressed in a baculovirus system.     

Hira基因与发育：从酵母到人类

Hir/Hira基因产物HIR/HIRA为组蛋白的伴侣蛋白,最先作为组蛋白基因表达的一种负调节因子从酵母中被鉴定出来。现已证实,HIRA包含一组保守的蛋白家族,广泛存在于低等真核生物、无脊椎动物和脊椎动物等多种生物体当中,为生命发育所必需。Hir/Hira基因功能突变对酵母以及高等真核生物的发育都有非常严重的影响。结合研究组的工作,综述了组蛋白调节基因Hir/Hira在不同生物体发育过程中的作用,以及该领域的研究方向之一 ¾ HIRA作用机理的最新进展。     

Regulation of meiotic recombination and prophase I progression in mammals.

Meiosis is the process by which diploid germ cells divide to produce haploid gametes for sexual reproduction. The process is highly conserved in eukaryotes, however the recent availability of mouse models for meiotic recombination has revealed surprising regulatory differences between simple unicellular organisms and those with increasingly complex genomes. Moreover, in these higher eukaryotes, the intervention of physiological and sex-specific factors may also influence how meiotic recombination and progression are monitored and regulated. This review will focus on the recent studies involving mouse mutants for meiosis, and will highlight important differences between traditional model systems for meiosis (such as yeast) and those involving more complex cellular, physiological and genetic criteria.