Fission yeast cdc31p is a component of the half-bridge and controls SPB duplication

The fission yeast spindle pole body (SPB) is a nucleus-associated organelle that duplicates once each cell cycle during interphase. Duplicated SPBs serve as the poles of an intranuclear mitotic spindle after their insertion into the nuclear envelope in mitosis (Ding et al., Mol. Biol. Cell 8, 1461-1479). Here, we report the identification and characterization of Schizosaccharomyces pombe cdc31p, a member of the conserved calcium-binding centrin/CDC31 family. Immunofluorescence and immunoelectron microscopy show that cdc31p is a SPB component localized at the half-bridge structure of the SPB. cdc31 is an essential gene and Deltacdc31 cells and cdc31 conditional mutant cells arrest in mitosis with a monopolar mitotic spindle organized from a single SPB. EM analysis demonstrates that mutant cdc31 cells fail to duplicate the SPB. In addition, cdc31p exhibits genetic interactions with the SPB component sad1p and is required for sad1p localization. Finally, cdc31 mutant can undergo single or multiple rounds of septation before the exit from mitosis, suggesting that cdc31p activity or SPB duplication may be required for the proper coordination between the exit from mitosis and the initiation of septation.     

The Bbp1p-Mps2p complex connects the SPB to the nuclear envelope and is essential for SPB duplication

In budding yeast, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope via its central plaque structure. Here, we describe the identification of BBP1 in a suppressor screen with a conditional lethal allele of SPC29. Bbp1p was detected at the central plaque periphery of the SPB and bbp1-1 cells were found to be defective in SPB duplication. bbp1-1 cells extend their satellite into a duplication plaque like wild-type cells; however, this duplication plaque then fails to insert properly into the nuclear envelope and does not assemble a functional inner plaque. This function in SPB duplication is probably fulfilled by a stable complex of Bbp1p and Mps2p, a nuclear envelope protein that is also essential for duplication plaque insertion. In addition, we found that Bbp1p interacts with Spc29p and the half-bridge component Kar1p. These interactions are likely to play a role in connecting the SPB with the nuclear envelope and the central plaque with the half-bridge.     

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae.

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.     

Functional interaction between BLM helicase and 53BP1 in a Chk1-mediated pathway during S-phase arrest

Bloom's syndrome is a rare autosomal recessive genetic disorder characterized by chromosomal aberrations, genetic instability, and cancer predisposition, all of which may be the result of abnormal signal transduction during DNA damage recognition. Here, we show that BLM is an intermediate responder to stalled DNA replication forks. BLM colocalized and physically interacted with the DNA damage response proteins 53BP1 and H2AX. Although BLM facilitated physical interaction between p53 and 53BP1, 53BP1 was required for efficient accumulation of both BLM and p53 at the sites of stalled replication. The accumulation of BLM/53BP1 foci and the physical interaction between them was independent of gamma-H2AX. The active Chk1 kinase was essential for both the accurate focal colocalization of 53BP1 with BLM and the consequent stabilization of BLM. Once the ATR/Chk1- and 53BP1-mediated signal from replicational stress is received, BLM functions in multiple downstream repair processes, thereby fulfilling its role as a caretaker tumor suppressor.     

Preferential duplication in the sparse part of yeast protein interaction network

Gene duplication is an important mechanism driving the evolution of biomolecular network. Thus, it is expected that there should be a strong relationship between a gene's duplicability and the interactions of its protein product with other proteins in the network. We studied this question in the context of the protein interaction network (PIN) of Saccharomyces cerevisiae. We found that duplicates have, on average, significantly lower clustering coefficient (CC) than singletons, and the proportion of duplicates (PD) decreases steadily with CC. Furthermore, using functional annotation data, we observed a strong negative correlation between PD and the mean CC for functional categories. By partitioning the network into modules and assigning each protein a modularity measure Q(n), we found that CC of a protein is a reflection of its modularity. Moreover, the core components of complexes identified in a recent high-throughput experiment, characterized by high CC, have lower PD than that of the attachments. Subsequently, 2 types of hub were identified by their degree, CC and Q(n). Although PD of intramodular hubs is much less than the network average, PD of intermodular hubs is comparable to, or even higher than, the network average. Our results suggest that high CC, and thus high modularity, pose strong evolutionary constraints on gene duplicability, and gene duplication prefers to happen in the sparse part of PINs.     

Functional evolution of the yeast protein interaction network

Protein interactions are central to most biological processes. We investigated the dynamics of emergence of the protein interaction network of Saccharomyces cerevisiae by mapping origins of proteins on an evolutionary tree. We demonstrate that evolutionary periods are characterized by distinct connectivity levels of the emerging proteins. We found that the most-connected group of proteins dates to the eukaryotic radiation, and the more ancient group of pre-eukaryotic proteins is less connected. We show that functional classes have different average connectivity levels and that the time of emergence of these functional classes parallels the observed connectivity variation in evolution. We take these findings as evidence that the evolution of function might be the reason for the differences in connectivity throughout evolutionary time. We propose that the understanding of the mechanisms that generate the scale-free protein interaction network, and possibly other biological networks, requires consideration of protein function.     

Functional sites of interaction between release factor RF1 and the ribosome

Translational release factors decipher stop codons in mRNA and activate hydrolysis of peptidyl-tRNA in the ribosome during translation termination. The mechanisms of these fundamental processes are unknown. Here we have mapped the interaction of bacterial release factor RF1 with the ribosome by directed hydroxyl radical probing. These experiments identified conserved domains of RF1 that interact with the decoding site of the 30S ribosomal subunit and the peptidyl transferase site of the 50S ribosomal subunit. RF1 interacts with a binding pocket formed between the ribosomal subunits that is also the interaction surface of elongation factor EF-G and aminoacyl-tRNA bound to the A site. These results provide a basis for understanding the mechanism of stop codon recognition coupled to hydrolysis of peptidyl-tRNA, mediated by a protein release factor.     

Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway.

Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature. We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response. cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor. On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants. These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein. The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response. A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle. A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein. Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.     

The identification of a functional interaction between PKC and topoisomerase II

Topoisomerase II plays an essential role in the segregation of chromosomes during cell division. It is also a major component of the nuclear matrix. Proteins that interact with and regulate this essential enzyme are of great interest. To investigate the role of proteins interacting with the N-terminal domain of the Saccharomyces cerevisiae topoisomerase II, we used a yeast two-hybrid protein interaction screen. We identified an interaction between the catalytic domain of the yeast protein kinase 1 enzyme (Pkc1) and the N-terminal domain of the S. cerevisiae topoisomerase II. The S. cerevisiae Pkc1 is the homologue of the mammalian calcium dependent PKC.     

Molecular interaction between fukutin and POMGnT1 in the glycosylation pathway of alpha-dystroglycan

The recent identification of mutations in genes encoding demonstrated or putative glycosyltransferases has revealed a novel mechanism for congenital muscular dystrophy. Hypoglycosylated alpha-dystroglycan (alpha-DG) is commonly seen in Fukuyama-type congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), Walker-Warburg syndrome (WWS), and Large(myd) mice. POMGnT1 and POMTs, the gene products responsible for MEB and WWS, respectively, synthesize unique O-mannose sugar chains on alpha-DG. The function of fukutin, the gene product responsible for FCMD, remains undetermined. Here we show that fukutin co-localizes with POMGnT1 in the Golgi apparatus. Direct interaction between fukutin and POMGnT1 was confirmed by co-immunoprecipitation and two-hybrid analyses. The transmembrane region of fukutin mediates its localization to the Golgi and participates in the interaction with POMGnT1. Y371C, a missense mutation found in FCMD, retains fukutin in the ER and also redirects POMGnT1 to the ER. Finally, we demonstrate reduced POMGnT1 enzymatic activity in transgenic knock-in mice carrying the retrotransposal insertion in the fukutin gene, the prevalent mutation in FCMD. From these findings, we propose that fukutin forms a complex with POMGnT1 and may modulate its enzymatic activity.     

Functional interaction between the herpes simplex-1 DNA polymerase and UL42 protein

The herpes simplex virus 1 (HSV-1) UL42 protein, one of seven herpes-encoded polypeptides that are required for the replication of the HSV-1 genome, is found in a 1:1 complex with the HSV-1 DNA polymerase (Crute, J. J., and Lehman, I. R. (1989) J. Biol. Chem. 264, 19266-19270). To obtain herpes DNA polymerase free of UL42 protein, we have cloned and overexpressed the Pol gene in a recombinant baculovirus vector and purified the recombinant DNA polymerase to near homogeneity. Replication of singly primed M13mp18 single-stranded DNA by the recombinant enzyme in the presence of the herpes encoded single-stranded DNA-binding protein ICP8 yields in addition to some full-length product a distribution of intermediate length products by a quasi-processive mode of deoxynucleotide polymerization. Addition of the purified UL42 protein results in completely processive polymerization and the generation of full-length products. Similar processivity is observed with the HSV-1 DNA polymerase purified from herpes-infected Vero cells. Processive DNA replication by the DNA polymerase isolated from HSV-1-infected Vero cells or the recombinant DNA polymerase-UL42 protein complex requires that the single-stranded DNA be coated with saturating levels of ICP8. ICP8 which binds single-stranded DNA in a highly cooperative manner is presumably required to melt out regions of secondary structure in the single-stranded DNA template, thereby potentiating the processivity enhancing action of the UL42 protein.     

Functional and direct interaction between the RNA binding protein HuD and active Akt1

The RNA binding protein HuD plays essential roles in neuronal development and plasticity. We have previously shown that HuD stimulates translation. Key for this enhancer function is the linker region and the poly(A) binding domain of HuD that are also critical for its function in neurite outgrowth. Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1. We identify that the linker region of HuD is required for this interaction. We also show by using chimeric mutants of HuD and HuR, which contain the reciprocal linker between RNA-binding domain 2 (RBD2) and RBD3, respectively, and by overexpressing a dominant negative mutant of Akt1 that the HuD-Akt1 interaction is functionally important, as it is required for the induction of neurite outgrowth in PC12 cells. These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth. These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.     

Functional clustering of yeast proteins from the protein-protein interaction network

Background  

The abundant data available for protein interaction networks have not yet been fully understood. New types of analyses are needed to reveal organizational principles of these networks to investigate the details of functional and regulatory clusters of proteins.     

Functional redundancy and new roles for genes of the autonomous floral-promotion pathway

The early-flowering habit of rapid-cycling accessions of Arabidopsis (Arabidopsis thaliana) is, in part, due to the genes of the autonomous floral-promotion pathway (AP). The AP promotes flowering by repressing expression of the floral inhibitor FLOWERING LOCUS C (FLC). AP mutants are therefore late flowering due to elevated levels of FLC, and this late-flowering phenotype is eliminated by loss-of-function mutations in FLC. To further investigate the role of the AP, we created a series of double mutants. In contrast to the phenotypes of single mutants, which are largely limited to delayed flowering, a subset of AP double mutants show a range of defects in growth and development. These phenotypes include reduced size, chlorophyll content, growth rate, and fertility. Unlike the effects of the AP on flowering time, these phenotypes are FLC independent. Recent work has also shown that two AP genes, FCA and FPA, are required for the repression and, in some cases, proper DNA methylation of two transposons. We show that similar effects are seen for all AP genes tested. Microarray analysis of gene expression in AP single and double mutants, however, suggests that the AP is not likely to play a broad role in the repression of gene expression through DNA methylation: very few of the genes that have been reported to be up-regulated in DNA methylation mutants are misexpressed in AP mutants. Together, these data indicate that the genes of the AP play important and sometimes functionally redundant roles in aspects of development in addition to flowering time.     

Functional binding interaction identified between the axonal CAM L1 and members of the ERM family

A yeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane-cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM-actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis.