Biomedical subjects

Matthias Peter

Publications and source records attributed to Matthias Peter.

At least 19 recordsLinked to original sources

PubMed2006

The F-box protein Dia2 overcomes replication impedance to promote genome stability in Saccharomyces cerevisiae.

The maintenance of DNA replication fork stability under conditions of DNA damage and at natural replication pause sites is essential for genome stability. Here, we describe a novel role for the F-box protein Dia2 in promoting genome stability in the budding yeast Saccharomyces cerevisiae. Like most other F-box proteins, Dia2 forms a Skp1-Cdc53/Cullin-F-box (SCF) E3 ubiquitin-ligase complex. Systematic analysis of genetic interactions between dia2Delta and approximately 4400 viable gene deletion mutants revealed synthetic lethal/synthetic sick interactions with a broad spectrum of DNA replication, recombination, checkpoint, and chromatin-remodeling pathways. dia2Delta strains exhibit constitutive activation of the checkpoint kinase Rad53 and elevated counts of endogenous DNA repair foci and are unable to overcome MMS-induced replicative stress. Notably, dia2Delta strains display a high rate of gross chromosomal rearrangements (GCRs) that involve the rDNA locus and an increase in extrachromosomal rDNA circle (ERC) formation, consistent with an observed enrichment of Dia2 in the nucleolus. These results suggest that Dia2 is essential for stable passage of replication forks through regions of damaged DNA and natural fragile regions, particularly the replication fork barrier (RFB) of rDNA repeat loci. We propose that the SCFDia2 ubiquitin ligase serves to modify or degrade protein substrates that would otherwise impede the replication fork in problematic regions of the genome.

Cell Cycle↗

PubMed2006

The cullin Rtt101p promotes replication fork progression through damaged DNA and natural pause sites.

Accurate and complete DNA replication is fundamental to maintain genome integrity. While the mechanisms and underlying machinery required to duplicate bulk genomic DNA are beginning to emerge, little is known about how cells replicate through damaged areas and special chromosomal regions such as telomeres, centromeres, and highly transcribed loci . Here, we have investigated the role of the yeast cullin Rtt101p in this process. We show that rtt101Delta cells accumulate spontaneous DNA damage and exhibit a G(2)/M delay, even though they are fully proficient to detect and repair chromosome breaks. Viability of rtt101Delta mutants depends on Rrm3p, a DNA helicase involved in displacing proteinaceous complexes at programmed pause sites . Moreover, rtt101Delta cells show hyperrecombination at forks arrested at replication fork barriers (RFBs) of ribosomal DNA. Finally, rtt101Delta mutants are sensitive to fork arrest induced by DNA alkylation, but not by nucleotide depletion. We therefore propose that the cullin Rtt101p promotes fork progression through obstacles such as DNA lesions or tightly bound protein-DNA complexes via a new mechanism involving ubiquitin-conjugation.

Cullin Proteins↗

PubMed2006

Scaffold proteins in MAP kinase signaling: more than simple passive activating platforms.

Due to the central position of scaffold proteins in numerous signaling networks, especially in MAPK pathways, considerable efforts have been made to identify new scaffolds and to characterize their function and regulation. Most of our knowledge stems from studies of yeast MAPK scaffolds, but the identification of such scaffolds in higher eukaryotes provided a new dimension to this field and led to exciting and promising new insights into the regulation of MAPK signaling. In this review, we shortly summarize the well-established basic functions of scaffolds in yeast and highlight concepts emerging from recent studies in yeast and higher eukaryotes. In particular, we discuss how scaffolds may actively influence MAPK signaling by inducing conformational changes of bound kinases or substrates, by controlling the localization of activated MAPK and the extent and output of MAPK activation, and by modulating MAPK kinetics through the recruitment of phosphatases or ubiquitin-ligases. Finally, we summarize the current knowledge of scaffold regulation, and how these events may be functionally important for MAPK signaling.

Allosteric Regulation↗

PubMed2005

Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis.

Translesion synthesis (TLS) is the major pathway by which mammalian cells replicate across DNA lesions. Upon DNA damage, ubiquitination of proliferating cell nuclear antigen (PCNA) induces bypass of the lesion by directing the replication machinery into the TLS pathway. Yet, how this modification is recognized and interpreted in the cell remains unclear. Here we describe the identification of two ubiquitin (Ub)-binding domains (UBM and UBZ), which are evolutionarily conserved in all Y-family TLS polymerases (pols). These domains are required for binding of poleta and poliota to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of poleta is essential to efficiently restore a normal response to ultraviolet irradiation in xeroderma pigmentosum variant (XP-V) fibroblasts. Our results indicate that Ub-binding domains of Y-family polymerases play crucial regulatory roles in TLS.

Amino Acid Motifs↗

PubMed2005

The BTB protein MEL-26 promotes cytokinesis in C. elegans by a CUL-3-independent mechanism.

BACKGROUND: The initiation of a cleavage furrow is essential to separate cells during cytokinesis, but little is known about the mechanisms controlling this actin-driven process. Previous studies in C. elegans embryos revealed that inactivation of the CUL-3-based E3 ligase activator rfl-1 results in an aberrant microtubule network, ectopic furrowing during pronuclear migration, and defects during cytokinesis. RESULTS: Here, we show that MEL-26, a substrate-specific adaptor of the CUL-3-based E3 ligase, is required for efficient cell separation and cleavage furrow ingression during the C. elegans early mitotic divisions. Loss of MEL-26 function leads to delayed onset and slow ingression of cytokinesis furrows that frequently regress. Conversely, increased levels of MEL-26 in cul-3(RNAi) and rfl-1 mutant embryos cause a hypercontractile cortex, with several simultaneously ingressing furrows during pronuclear migration. MEL-26 accumulates at cleavage furrows and binds the actin-interacting protein POD-1. Importantly, POD-1 is not a substrate of the MEL-26/CUL-3 ligase but is required to localize MEL-26 to the cortex. CONCLUSIONS: Our results suggest that MEL-26 not only acts as a substrate-specific adaptor within the MEL-26/CUL-3 complex, but also promotes cytokinesis by a CUL-3- and microtubule-independent mechanism.

Adaptor Proteins, Signal Transducing↗

PubMed2005

The Bud14p-Glc7p complex functions as a cortical regulator of dynein in budding yeast.

Regulated interactions between microtubules (MTs) and the cell cortex control MT dynamics and position the mitotic spindle. In eukaryotic cells, the adenomatous polyposis coli/Kar9p and dynein/dynactin pathways are involved in guiding MT plus ends and MT sliding along the cortex, respectively. Here we identify Bud14p as a novel cortical activator of the dynein/dynactin complex in budding yeast. Bud14p accumulates at sites of polarized growth and the mother-bud neck during cytokinesis. The localization to bud and shmoo tips requires an intact actin cytoskeleton and the kelch-domain-containing proteins Kel1p and Kel2p. While cells lacking Bud14p function fail to stabilize the pre-anaphase spindle at the mother-bud neck, overexpression of Bud14p is toxic and leads to elongated astral MTs and increased dynein-dependent sliding along the cell cortex. Bud14p physically interacts with the type-I phosphatase Glc7p, and localizes Glc7p to the bud cortex. Importantly, the formation of Bud14p-Glc7p complexes is necessary to regulate MT dynamics at the cortex. Taken together, our results suggest that Bud14p functions as a regulatory subunit of the Glc7p type-I phosphatase to stabilize MT interactions specifically at sites of polarized growth.

Actin Cytoskeleton↗

PubMed2005

The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae.

SCF-type E3 ubiquitin ligases are multi-protein complexes required for polyubiquitination and subsequent degradation of target proteins by the 26S proteasome. Cullins, together with the RING-finger protein Rbx1, form the catalytic core of the ligase, and recruit the substrate-recognition module. Cycles of covalent modification of cullins by the ubiquitin-like molecule Nedd8 (neddylation) and removal of Nedd8 by the COP9 signalosome (deneddylation) positively regulate E3 ligase activity. Here we report the identification and analysis of a widely conserved protein that is required for cullin neddylation in the nematode Caenorhabditis elegans and the yeast Saccharomyces cerevisiae. C. elegans DCN-1 and S. cerevisiae Dcn1p (defective in cullin neddylation) are characterized by a novel UBA-like ubiquitin-binding domain and a DUF298 domain of unknown function. Consistent with their requirements for neddylation, DCN-1 and Dcn1p directly bind Nedd8 and physically associate with cullins in both species. Moreover, overexpression of Dcn1p in yeast results in the accumulation of Nedd8-modified cullin Cdc53p. Both in vivo and in vitro experiments indicate that Dcn1p does not inhibit deneddylation of Cdc53p by the COP9 signalosome, but greatly increases the kinetics of the neddylation reaction.

Adenosine Triphosphatases↗

PubMed2005

Degradation of Hof1 by SCF(Grr1) is important for actomyosin contraction during cytokinesis in yeast.

SCF-type (SCF: Skp1-Cullin-F-box protein complex) E3 ligases regulate ubiquitin-dependent degradation of many cell cycle regulators, mainly at the G1/S transition. Here, we show that SCF(Grr1) functions during cytokinesis by degrading the PCH protein Hof1. While Hof1 is required early in mitosis to assemble a functional actomyosin ring, it is specifically degraded late in mitosis and remains unstable during the entire G1 phase of the cell cycle. Degradation of Hof1 depends on its PEST motif and a functional 26S proteasome. Interestingly, degradation of Hof1 is independent of APC(Cdh1), but instead requires the SCF(Grr1) E3 ligase. Grr1 is recruited to the mother-bud neck region after activation of the mitotic-exit network, and interacts with Hof1 in a PEST motif-dependent manner. Our results also show that downregulation of Hof1 at the end of mitosis is necessary to allow efficient contraction of the actomyosin ring and cell separation during cytokinesis. SCF(Grr1)-mediated degradation of Hof1 may thus represent a novel mechanism to couple exit from mitosis with initiation of cytokinesis.

Actomyosin↗

PubMed2005

Control of cell polarity in response to intra- and extracellular signals in budding yeast.

Budding yeast serves as a powerful genetic model organism for studying the molecular mechanisms of cell polarity in single cells. Like other polarized eukaryotic cells, yeast cells possess polarity programs that regulate where they grow and divide. Establishment of a site of cell polarity may be conceptualized in several stages. First, cells mark a specific location at the cell surface for polarized cell growth and cell division. To define these sites, cells use intrinsic cues present in the cell or landmarks determined by extracellular signals such as morphogens. Second, these landmark proteins then recruit or activate polarity establishment proteins including small GTPases and their regulators. Positive and negative feedback mechanisms are required to transform these site-selection processes into a stable axis of polarity. Finally, these locally activated GTPase modules recruit and activate proteins that organize the actin cytoskeleton and cell growth. In this short review, we describe molecular pathways required to establish oriented cell polarity, and emphasize recent advances in defining positive and negative feedback mechanisms that together may translate an initially weak symmetry-breaking signal into a robust axis of polarity.

Cell Polarity↗

PubMed2004

Cullin-based ubiquitin ligases: Cul3-BTB complexes join the family.

Cullin-based E3 ligases target substrates for ubiquitin-dependent degradation by the 26S proteasome. The SCF (Skp1-Cul1-F-box) and ECS (ElonginC-Cul2-SOCS box) complexes are so far the best-characterized cullin-based ligases. Their atomic structure has been solved recently, and several substrates have been described in different organisms. In addition to Cul1 and Cul2, higher eucaryotic genomes encode for three other cullins: Cul3, Cul4, and Cul5. Recent results have shed light on the molecular composition and function of Cul3-based E3 ligases. In these complexes, BTB-domain-containing proteins may bridge the cullin to the substrate in a single polypeptide, while Skp1/F-box or ElonginC/SOCS heterodimers fulfill this function in the SCF and ECS complexes. BTB-containing proteins are evolutionary conserved and involved in diverse biological processes, but their function has not previously been linked to ubiquitin-dependent degradation. In this review, we present these new findings and compare the composition of Cul3-based ligases to the well-defined SCF and ECS ligases.

Animals↗

PubMed2004

Insight into molecular interactions between two PB1 domains.

Specific protein-protein interactions play crucial roles in the regulation of any biological process. Recently, a new protein-protein interaction domain termed PB1 (Phox and Bem1) was identified, which is conserved throughout evolution and present in diverse proteins functioning in signal transduction, cell polarity and survival. Here, we investigated the structure and molecular interactions of the PB1 heterodimer complex composed of the PB1 domains of the yeast proteins Bem1 and Cdc24. A structural model of the Cdc24 PB1 was built by homology modeling and molecular dynamics simulations, and experimentally validated by 15N nuclear Overhauser effect spectroscopy (NOESY)-heteronuclear single quantum coherence (HSQC) analysis. Residues at the interface of the complex for both proteins were identified by NMR titration experiments. A model of the heterodimer was obtained by docking of the two PB1 domains with HADDOCK, which applies ambiguous interaction restraints on residues at the interface to drive the docking procedure. The refined model was validated by site-directed mutagenesis on both Bem1 and Cdc24. Finally, the docking was repeated from the newly published NMR structure of Cdc24, allowing us to assess the performance of homology-based docking. Our results provide insight into the molecular structure of the Bem1-Cdc24 PB1-mediated heterodimer, which allowed identification of critical residues at the binding interface.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Site-specific regulation of the GEF Cdc24p by the scaffold protein Far1p during yeast mating.

Receptor-mediated cell polarization via heterotrimeric G-proteins induces cytoskeletal rearrangements in a variety of organisms. In yeast, Far1p is required for orienting cell growth towards the mating partner by linking activated Gbetagamma to the guanine-nucleotide exchange factor (GEF) Cdc24p, which activates the Rho-type GTPase Cdc42p. Here we investigated the role of Far1p in the regulation of Cdc24p in vivo. Using time-lapse microscopy of mating cells and artificial membrane targeting of Far1p, we show that Far1p is necessary and sufficient to recruit Cdc24p to the plasma membrane. Wild-type Far1p contains a PH-like domain, which is required for its membrane localization in vivo. Interestingly, expression of membrane-targeted Far1p causes toxicity, most likely by activating Cdc42p uniformly at the cell cortex. The ability of full-length Far1p to function as an activator of Cdc24p in vivo requires its interaction with Cdc24p and Gbetagamma. Our results imply that Gbetagamma not only targets Far1p to the correct site but may also trigger a conformational change in Far1p that is required for its ability to activate Cdc24p in vivo.

Amino Acid Sequence↗

PubMed2004

The nucleotide exchange factor Cdc24p may be regulated by auto-inhibition.

Site-specific activation of the Rho-type GTPase Cdc42p by its guanine-nucleotide exchange factor (GEF) Cdc24p is critical for the establishment of cell polarity. Here we show that binding of Cdc24p to the small GTPase Rsr1p/Bud1p is required for its recruitment to the incipient bud site. Rsr1p/Bud1p binds to the CH-domain of Cdc24p, which is essential for its function in vivo. We have identified a cdc24-mutant allele, which is specifically defective for bud-site selection. Our results suggest that Cdc24p is auto-inhibited by an intramolecular interaction with its carboxy-terminal PB1-domain. Rsr1p/Bud1p appears to activate the GEF activity of Cdc24p in vivo, possibly by triggering a conformational change that dissociates the PB1-domain from its intramolecular binding site. Genetic experiments suggest that Bem1p functions as a positive regulator of Cdc24p by binding to the PB1-domain of Cdc24p, thereby preventing its re-binding to the intramolecular inhibitory site. Taken together, our results support a two-step molecular mechanism for the site-specific activation of Cdc24p, which involves Rsr1p/Bud1p and the adaptor protein Bem1p.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Global mapping of the yeast genetic interaction network.

A genetic interaction network containing approximately 1000 genes and approximately 4000 interactions was mapped by crossing mutations in 132 different query genes into a set of approximately 4700 viable gene yeast deletion mutants and scoring the double mutant progeny for fitness defects. Network connectivity was predictive of function because interactions often occurred among functionally related genes, and similar patterns of interactions tended to identify components of the same pathway. The genetic network exhibited dense local neighborhoods; therefore, the position of a gene on a partially mapped network is predictive of other genetic interactions. Because digenic interactions are common in yeast, similar networks may underlie the complex genetics associated with inherited phenotypes in other organisms.

Amino Acid Sequence↗

PubMed2004

A genome-wide screen in Saccharomyces cerevisiae reveals altered transport as a mechanism of resistance to the anticancer drug bleomycin.

The potent DNA damaging agent bleomycin (BLM) is highly effective for treating various cancers, although, in certain individuals, the development of cellular resistance to the drug can severely diminish its antineoplastic properties. We performed two independent genome-wide screens using a Saccharomyces cerevisiae mutant collection to isolate variants exhibiting either sensitivity or resistance to BLM. This procedure reproducibly identified a relatively large collection of 231 BLM-hypersensitive mutants, representing genes belonging to diverse functional groups. In contrast, only five BLM-resistant mutants could be recovered by our screens. Among these latter mutants, three were deleted for genes involved in plasma membrane transport, including the L-carnitine transporter Agp2, as well as the kinases Ptk2 and Sky1, which are involved in regulating polyamine transport. We further showed that Agp2 acts as a transporter of BLM and that overexpression of this transporter significantly enhances BLM-induced cell killing. Our data strongly implicate membrane transport as a key determinant in BLM resistance in yeast. This finding is critical, given that very little is known about BLM transport in human cells. Indeed, characterization of analogous mechanisms in humans may ultimately lead to enhancement of the antitumor properties of BLM.

Amino Acid Transport Systems↗

PubMed2004

The GTPase-activating enzyme Gyp1p is required for recycling of internalized membrane material by inactivation of the Rab/Ypt GTPase Ypt1p.

Rab/Ypt GTPases are key regulators of membrane trafficking and together with SNARE proteins mediate selective fusion of vesicles with target compartments. A family of GTPase-activating enzymes (GAPs) specific for Rab/Ypt GTPases has been discovered, but little is known about their function and substrate specificity in vivo. Here we show that the GAP activity of Gyp1p, a yeast member of this family, is specifically required for recycling of the SNARE Snc1p and the membrane dye FM4-64, implying that inactivation of a Rab/Ypt GTPase may be necessary for recycling of membrane material. Interestingly, recycling of GFP-Snc1p in gyp1 Delta cells is partially restored by reducing the activity of Ypt1p. Moreover, GFP-Snc1p accumulated intracellularly in wild-type cells expressing a GTP-locked, mutant form of Ypt1p (Ypt1p-Q67L), suggesting that GTP hydrolysis of Ypt1p is essential for recycling. Ypt6p is known to be required for the fusion of recycling vesicles to the late Golgi compartment. Interestingly, the deletions of GYP1 and YPT6 were synthetic lethal, raising the possibility that at least two distinct pathways are involved in recycling of membrane material.

Amino Acid Sequence↗

PubMed2004

Revealing protein dynamics by photobleaching techniques.

Green fluorescent proteins (GFPs) are widely used tools to visualize proteins and study their intracellular distribution. One feature of working with GFP variants, photobleaching, has recently been combined with an older technique known as fluorescence recovery after photobleaching (FRAP) to study protein kinetics in vivo. During photobleaching, fluorochromes get destroyed irreversibly by repeated excitation with an intensive light source. When the photobleaching is applied to a restricted area or structure, recovery of fluorescence will be the result of active or passive diffusion from fluorescent molecules from unbleached surrounding areas. Fluorescence loss in photobleaching (FLIP) is a variant of FRAP where an area is bleached, and loss of fluorescence in surrounding areas is observed. FLIP can be used to study the dynamics of different pools of a protein or can show how a protein diffuses, or is transported through a cell or cellular structure. Here, we discuss these photobleaching fluorescent imaging techniques, illustrated with examples of these techniques applied to proteins of the Saccharomyces cerevisiae pheromone response MAPK pathway.

Escherichia coli↗

PubMed2003

Control of nutrient-sensitive transcription programs by the unconventional prefoldin URI.

Prefoldins (PFDs) are members of a recently identified, small-molecular weight protein family able to assemble into molecular chaperone complexes. Here we describe an unusually large member of this family, termed URI, that forms complexes with other small-molecular weight PFDs and with RPB5, a shared subunit of all three RNA polymerases. Functional analysis of the yeast and human orthologs of URI revealed that both are targets of nutrient signaling and participate in gene expression controlled by the TOR kinase. Thus, URI is a component of a signaling pathway that coordinates nutrient availability with gene expression.

Amino Acid Sequence↗