Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.     

Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.     

Use of peptides from p21 (Waf1/Cip1) to investigate PCNA function in Xenopus egg extracts

Cell-free systems derived from unfertilized Xenopus eggs have been particularly informative in the study of the regulation and biochemistry of DNA replication. We have developed a Xenopus-based system to analyze proliferating cell nuclear antigen (PCNA)-specific effects on the functional properties of egg extracts. To do this, we have coupled peptides derived from p21 (Waf1/Cip1) to beads and used these to deplete PCNA from Xenopus egg extracts. The effect on various aspects of DNA replication can be analyzed after the readdition of PCNA and other purified proteins. Using this system, we have shown that replication of single-stranded M13 DNA is entirely dependent upon PCNA. By adding exogenous T7 DNA polymerase to PCNA-depleted extracts, we have uncoupled processive DNA replication from PCNA activity and so created an experimental system to analyze the dependence of postreplicative processes on PCNA function. We have shown that successful chromatin assembly is specifically dependent on PCNA. However, systems for analyzing the far more complex mechanisms required for the replication of nuclear double-stranded DNA have proved so far to be refractory to specific PCNA depletion.     

Prediction of protein tertiary structure using PROFESY, a novel method based on fragment assembly and conformational space annealing

A novel method for ab initio prediction of protein tertiary structures, PROFESY (PROFile Enumerating SYstem), is proposed. This method utilizes the secondary structure prediction information of a query sequence and the fragment assembly procedure based on global optimization. Fifteen-residue-long fragment libraries are constructed using the secondary structure prediction method PREDICT, and fragments in these libraries are assembled to generate full-length chains of a query protein. Tertiary structures of 50 to 100 conformations are obtained by minimizing an energy function for proteins, using the conformational space annealing method that enables one to sample diverse low-lying local minima of the energy. We apply PROFESY for benchmark tests to proteins with known structures to demonstrate its feasibility. In addition, we participated in CASP5 and applied PROFESY to four new-fold targets for blind prediction. The results are quite promising, despite the fact that PROFESY was in its early stages of development. In particular, PROFESY successfully provided us the best model-one structure for the target T0161.     

Assembly of the phagocyte NADPH oxidase

Stimulated phagocytes undergo a burst in respiration whereby molecular oxygen is converted to superoxide anion through the action of an NADPH-dependent oxidase. The multicomponent phagocyte oxidase is unassembled and inactive in resting cells but assembles at the plasma or phagosomal membrane upon phagocyte activation. Oxidase components include flavocytochrome b₅₅₈, an integral membrane heterodimer comprised of gp91phox and p22phox, and three cytosolic proteins, p47phox, p67phox, and Rac1 or Rac2, depending on the species and phagocytic cell. In a sense, the phagocyte oxidase is spatially regulated, with critical elements segregated in the membrane and cytosol but ready to undergo nearly immediate assembly and activation in response to stimulation. To achieve such spatial regulation, the individual components in the resting phagocyte adopt conformations that mask potentially interactive structural domains that might mediate productive intermolecular associations and oxidase assembly. In response to stimulation, post-translational modifications of the oxidase components release these constraints and thereby render potential interfaces accessible and interactive, resulting in translocation of the cytosolic elements to the membrane where the functional oxidase is assembled and active. This review summarizes data on the structural features of the phagocyte oxidase components and on the agonist-dependent conformational rearrangements that contribute to oxidase assembly and activation.     

Cell-free systems for capsid assembly of primate lentiviruses from three different lineages

We recently demonstrated that capsids from three main primate lentiviral lineages appear to form via a pathway of assembly intermediates in primate cells. Retroviral capsid assembly intermediates were initially identified and characterized using a cell-free system for assembly of immature HIV-1 capsids. Because cell-free capsid assembly systems are useful tools, we are interested in developing such systems for other primate lentiviruses besides HIV-1. Here we extend previous cell-free studies by showing that Gag proteins of HIV-2, from a second primate lentiviral lineage, progress from early intermediates to late intermediates and completed capsids over time. Additionally, we demonstrate that Gag proteins of SIVagm, from a third primate lentiviral lineage, associate with the cellular factor HP68 and complete assembly in this system. Therefore, cell-free systems reproduce assembly of Gag from three main primate lentiviral lineages, and can be used to compare mechanistic features of capsid assembly of genetically divergent primate lentiviruses.     

Structure,function and assembly of Photosystem II and its light-harvesting proteins

Photosystem II (PSII) is a multisubunit chlorophyll–protein complex that drives electron transfer from water to plastoquinone using energy derived from light. In green plants, the native form of PSII is surrounded by the light-harvesting complex (LHCII complex) and thus it is called the PSII–LHCII supercomplex. Over the past several years, understanding of the structure, function, and assembly of PSII and LHCII complexes has increased considerably. The unicellular green alga Chlamydomonas reinhardtii has been an excellent model organism to study PSII and LHCII complexes, because this organism grows heterotrophically and photoautotrophically and it is amenable to biochemical, genetic, molecular biological and recombinant DNA methodology. Here, the genes encoding and regulating components of the C. reinhardtii PSII–LHCII supercomplex have been thoroughly catalogued: they include 15 chloroplast and 20 nuclear structural genes as well as 13 nuclear genes coding for regulatory factors. This review discusses these molecular genetic data and presents an overview of the structure, function and assembly of PSII and LHCII complexes.     

Prp5 bridges U1 and U2 snRNPs and enables stable U2 snRNP association with intron RNA

Communication between U1 and U2 snRNPs is critical during pre-spliceosome assembly; yet, direct connections have not been observed. To investigate this assembly step, we focused on Prp5, an RNA-dependent ATPase of the DExD/H family. We identified homologs of Saccharomyces cerevisiae Prp5 in humans (hPrp5) and Schizosaccharomyces pombe (SpPrp5), and investigated their interactions and function. Depletion and reconstitution of SpPrp5 from extracts demonstrate that ATP binding and hydrolysis by Prp5 are required for pre-spliceosome complex A formation. hPrp5 and SpPrp5 are each physically associated with both U1 and U2 snRNPs; Prp5 contains distinct U1- and U2-interacting domains that are required for pre-spliceosome assembly; and, we observe a Prp5-associated U1/U2 complex in S. pombe. Together, these data are consistent with Prp5 being a bridge between U1 and U2 snRNPs at the time of pre-spliceosome formation.     

Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae

Mutations in SURF1, the human homologue of yeast SHY1, are responsible for Leigh's syndrome, a neuropathy associated with cytochrome oxidase (COX) deficiency. Previous studies of the yeast model of this disease showed that mutant forms of Mss51p, a translational activator of COX1 mRNA, partially rescue the COX deficiency of shy1 mutants by restoring normal synthesis of the mitochondrially encoded Cox1p subunit of COX. Here we present evidence showing that Cox1p synthesis is reduced in most COX mutants but is restored to that of wild type by the same mss51 mutation that suppresses shy1 mutants. An important exception is a null mutation in COX14, which by itself or in combination with other COX mutations does not affect Cox1p synthesis. Cox14p and Mss51p are shown to interact with newly synthesized Cox1p and with each other. We propose that the interaction of Mss51p and Cox14p with Cox1p to form a transient Cox14p-Cox1p-Mss51p complex functions to downregulate Cox1p synthesis. The release of Mss51p from the complex occurs at a downstream step in the assembly pathway, probably catalyzed by Shy1p.     

A CAF-1 dependent pool of HP1 during heterochromatin duplication

To investigate how the complex organization of heterochromatin is reproduced at each replication cycle, we examined the fate of HP1-rich pericentric domains in mouse cells. We find that replication occurs mainly at the surface of these domains where both PCNA and chromatin assembly factor 1 (CAF-1) are located. Pulse-chase experiments combined with high-resolution analysis and 3D modeling show that within 90 min newly replicated DNA become internalized inside the domain. Remarkably, during this time period, a specific subset of HP1 molecules (alpha and gamma) coinciding with CAF-1 and replicative sites is resistant to RNase treatment. Furthermore, these replication-associated HP1 molecules are detected in Suv39 knockout cells, which otherwise lack stable HP1 staining at pericentric heterochromatin. This replicative pool of HP1 molecules disappears completely following p150CAF-1 siRNA treatment. We conclude that during replication, the interaction of HP1 with p150CAF-1 is essential to promote delivery of HP1 molecules to heterochromatic sites, where they are subsequently retained by further interactions with methylated H3-K9 and RNA.