The ability of cells to make exact replicas of themselves is central to the life and development of complex organisms. Initial insights into the question of how cells divide came during the latter half of the 19th century when Walther Flemming visualized structures he called threads (which we now call chromosomes) and described how these threads change during cell multiplication, a process he called mitosis. Now, more than a century later, we have a molecular understanding of many of the cellular processes that Flemming observed. Indeed, major cytological events occurring during mitosis are known to constitute cell cycle transitions and are regulated by complex signal transduction pathways whose major components have been identified during the past decade. In this review, we describe recent efforts to understand how central components of this regulatory apparatus-cyclin-dependent kinases and the anaphasepromoting complex/cyclosome (APC/C)-control progression through the cell division cycle and how regulatory mechanisms impinge on the APC/C. The APC/C is the multisubunit ubiquitin ligase whose activity is precisely regulated to ensure the timely degradation of cyclins and other key cell cycle regulators in unperturbed cells and to respond to mitotic checkpoints that prevent their degradation. We pay particular attention to recent developments as excellent reviews are available from a few years ago (Morgan 1999; Zachariae and Nasmyth 1999). Cell cycle transitions: interplay between cyclin-dependent kinases and ubiquitin-mediated proteolysisThe primary task of the cell division cycle is to duplicate genetic information precisely through the process of DNA replication (S phase) and then to allocate this information equally to two daughter cells through mitosis. Inaccuracies in this process can be problematic. For example, cells that attempt to separate chromosomes that are incorrectly or incompletely duplicated are much more likely to incur fatal or irreparable damage as a result of either loss or gain of genetic information. Thus, a large number of signaling pathways collaborate to enforce order on cell division events.Cell cycle transitions are the primary mechanism used by the cell to establish the order and timing of cell cycle events. Such transitions occur when there is a change in the biochemical status of the cell division machinery. Early cell-fusion experiments showed that major cell cycle phases can be incompatible with one another. For example, when a G 2 cell is fused with an S-phase cell, the G 2 nucleus waits until the S-phase nucleus has completed replication before both nuclei enter mitosis synchronously (Rao and Johnson 1970). Thus, progression through G 2 into mitosis is incompatible with ongoing DNA synthesis. Through subsequent genetic and biochemical analysis, we now understand in general terms how these cell cycle dependencies are generated and controlled. Moreover, molecules that play key roles in defining particular cell cycle stages have been uncovered. One frequently used paradigm involves...

Inappropriate attachment/tension between chromosomal kinetochores and the kinetochore microtubules activates the spindle assembly checkpoint, which delays anaphase by blocking the ubiquitin-mediated degradation of securin/Pds1p by APC [Keywords: Anaphase-promoting complex; budding yeast; Cdc20; cell cycle] Supplemental material is available at http://www.genesdev.org.

Secl is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae. Two high copy suppressors of mutations in the Secl gene, SSOI and SS02, were recently identified that encode proteins of the syntaxin family. Syntain (a T-SNARE), together with SNAP-25 and synaptobrevin/VAMP (a T-and a V-SNARE, respectively), is thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Secl were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabdiis ekgans (UNC18). Based on the amino acid sequence alignment ofSecl, Rop, and UNC18, we have used a PCR-based approach to isolate a rat brain cDNA encoding a Secl homologue. The cDNA hybridizes to a 3.5-kb brain-specific mRNA by Northern blot analysis and encodes a protein of593 amino acids (rbSecl). Antibodies raised against a central portion of rbSecl recognize a 67.5-kDa protein in total homogenates of rat brain but not of nonneuronal tissues. When incubated with a Triton X-100 brain extract, rbSecl-glutathione S-transferase (GST) fusion protein, but not GST protein alone, specificaly interacts with syntaxin but not with SNAP-25 or synaptobrevin/VAMP. We conclude that the function of proteins of the Secl family in membrane fusion involves an interaction with a T-SNARE.Elucidation of the molecular mechanisms by which synaptic vesicles dock and fuse with the plasmalemma has recently been the focus of intense investigation (1). Increasing evidence suggests that molecular mechanisms of synaptic vesicle exocytosis are fundamentally similar to mechanisms that operate in all types of exocytosis and, more generally, in all membrane fusion events in the secretory and endocytic pathway (2).A set of components that appear to form the core of the docking and fusion machinery for synaptic vesicles has been identified. According to a recently proposed model, referred to as the SNARE hypothesis (3), the synaptic vesicle protein synaptobrevin/VAMP (referred to as a V-SNARE) (4,5) interacts with syntaxin (6) and SNAP-25 (referred to as T-SNARES) (7), two proteins localized in the plasmalemma (3,8). (VAMP is a vesicle-associated membrane protein; SNAP is a soluble N-ethylmaleimide-sensitive attachment protein fusion protein; and SNARE is a SNAP receptor.) Homologues of each of these proteins are present in yeast, and genetic analysis has demonstrated their participation in exocytosis (refs. 9-11; V. Bankaitis, personal communication). Furthermore, the crucial role of these proteins in neuronal exocytosis is emphasized by the demonstration that they are targets for the proteolytic action of clostridial neurotoxins, which are potent inhibitors of neurotransmitter release (12)(13)(14). Formation of the SNARE complex is then thought to be followed by the recruitment of several cytosolic factors required for fusion including N-ethylmaleimidesensitive fusion proteins, a-, /-, and y-SNAPs (3). Corresponding yeast homologues, which play a general role in membrane...

The precise order of molecular events during cell cycle progression depends upon ubiquitin-mediated proteolysis of cell cycle regulators. We demonstrated previously that Hsl1p, a protein kinase that inhibits the Swe1p protein kinase in a bud morphogenesis checkpoint, is targeted for ubiquitin-mediated turnover by the anaphase-promoting complex (APC). Here, we investigate regions of Hsl1p that are critical both for binding to the APC machinery and for APC-mediated degradation. We demonstrate that Hsl1p contains both a destruction box (D box) and a KEN box motif that are necessary for Hsl1p turnover with either APC Cdc20 or APC Cdh1. In coimmunoprecipitation studies, the D box of full-length Hsl1p was critical for association with Cdc20p, whereas the KEN box was important for association with Cdh1p.

Mss4 is a mammalian protein that was identified as a suppressor of a yeast secretory mutant harboring a mutation in the GTPase Sec4 and was found to stimulate GDP release from this protein. We have now performed a biochemical characterization of the Mss4 protein and examined the specificity of its association with mammalian GTPases. Mss4 is primarily a soluble protein with a widespread tissue distribution. Recombinant Mss4 binds GTPases present in tissue extracts, and by a gel overlay assay binds specifically Rab Rab10proteins. We further define the Mss4‐GTPase interaction to a subset of Rabs belonging to the same subfamily branch which include Rab1, Rab3, Rab8, Rab10, Sec4 and Ypt1 but not Rab2, Rab4, Rab5, Rab6, Rab9 and Rab11. Accordingly, Mss4 co‐precipitates from a brain extract with Rab3a but not Rab5. Mss4 only stimulates GDP release from, and the association of GTP gamma S with, this Rab subset. Recombinant Mss4 and Rab3a form a stable complex in solution that is dissociated with either GDP or GTP gamma S. Injection of Mss4 into the squid giant nerve terminal enhances neurotransmitter release. These results suggest that Mss4 behaves as a guanylnucleotide exchange factor (GEF) for a subset of Rabs to influence distinct vesicular transport steps along the secretory pathway.