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1.  Phosphoproteomic analyses reveal novel cross-modulation mechanisms between two signaling pathways in yeast 
Molecular Systems Biology  2014;10(12):767.
Cells respond to environmental stimuli via specialized signaling pathways. Concurrent stimuli trigger multiple pathways that integrate information, predominantly via protein phosphorylation. Budding yeast responds to NaCl and pheromone via two mitogen-activated protein kinase cascades, the high osmolarity, and the mating pathways, respectively. To investigate signal integration between these pathways, we quantified the time-resolved phosphorylation site dynamics after pathway co-stimulation. Using shotgun mass spectrometry, we quantified 2,536 phosphopeptides across 36 conditions. Our data indicate that NaCl and pheromone affect phosphorylation events within both pathways, which thus affect each other at more levels than anticipated, allowing for information exchange and signal integration. We observed a pheromone-induced down-regulation of Hog1 phosphorylation due to Gpd1, Ste20, Ptp2, Pbs2, and Ptc1. Distinct Ste20 and Pbs2 phosphosites responded differently to the two stimuli, suggesting these proteins as key mediators of the information exchange. A set of logic models was then used to assess the role of measured phosphopeptides in the crosstalk. Our results show that the integration of the response to different stimuli requires complex interconnections between signaling pathways.
PMCID: PMC4300490  PMID: 25492886
cell signaling network; crosstalk; HOG pathway; pheromone pathway; phosphoproteomics
2.  Structural and Biochemical Characterization of the Cop9 Signalosome CSN5/CSN6 Heterodimer 
PLoS ONE  2014;9(8):e105688.
The Cop9 signalosome complex (CSN) regulates the functional cycle of the major E3 ubiquitin ligase family, the cullin RING E3 ubiquitin ligases (CRLs). Activated CRLs are covalently modified by the ubiquitin-like protein Nedd8 (neural precursor cell expressed developmentally down-regulated protein 8). CSN serves an essential role in myriad cellular processes by reversing this modification through the isopeptidase activity of its CSN5 subunit. CSN5 alone is inactive due to an auto-inhibited conformation of its catalytic domain. Here we report the molecular basis of CSN5 catalytic domain activation and unravel a molecular hierarchy in CSN deneddylation activity. The association of CSN5 and CSN6 MPN (for Mpr1/Pad1 N-terminal) domains activates its isopeptidase activity. The CSN5/CSN6 module, however, is inefficient in CRL deneddylation, indicating a requirement of further elements in this reaction such as other CSN subunits. A hybrid molecular model of CSN5/CSN6 provides a structural framework to explain these functional observations. Docking this model into a published CSN electron density map and using distance constraints obtained from cross-linking coupled to mass-spectrometry, we find that the C-termini of the CSN subunits could form a helical bundle in the centre of the structure. They likely play a key scaffolding role in the spatial organization of CSN and precise positioning of the dimeric MPN catalytic core.
PMCID: PMC4140821  PMID: 25144743
3.  Early Steps in Autophagy Depend on Direct Phosphorylation of Atg9 by the Atg1 Kinase 
Molecular Cell  2014;53(3):471-483.
Bulk degradation of cytoplasmic material is mediated by a highly conserved intracellular trafficking pathway termed autophagy. This pathway is characterized by the formation of double-membrane vesicles termed autophagosomes engulfing the substrate and transporting it to the vacuole/lysosome for breakdown and recycling. The Atg1/ULK1 kinase is essential for this process; however, little is known about its targets and the means by which it controls autophagy. Here we have screened for Atg1 kinase substrates using consensus peptide arrays and identified three components of the autophagy machinery. The multimembrane-spanning protein Atg9 is a direct target of this kinase essential for autophagy. Phosphorylated Atg9 is then required for the efficient recruitment of Atg8 and Atg18 to the site of autophagosome formation and subsequent expansion of the isolation membrane, a prerequisite for a functioning autophagy pathway. These findings show that the Atg1 kinase acts early in autophagy by regulating the outgrowth of autophagosomal membranes.
Graphical Abstract
•The Atg1 kinase phosphorylation consensus was identified on peptide arrays•Atg9 is a direct target of the Atg1/ULK1 kinase in vitro and in vivo•Atg9 phosphorylation recruits Atg18 and Atg8 to the PAS•Atg9 phosphorylation is required for isolation membrane expansion/autophagy function
Autophagy function is pivotal to cell health. Papinski et al. identify the phosphorylation consensus of the central kinase in this pathway, Atg1. The autophagy-related protein Atg9 is a direct target of Atg1. Atg9 phosphorylation by Atg1 is required for autophagosome formation. This finding sheds light on how Atg1 controls autophagy.
PMCID: PMC3978657  PMID: 24440502
5.  Atg1 kinase regulates early and late steps during autophagy 
Autophagy  2013;9(2):249-251.
The notion that phosphorylation constitutes a major mechanism to induce autophagy was established 15 years ago when a conserved Atg1/ULK kinase family was identified as an essential component of the autophagy machinery. The key observation was that starved atg1Δ cells lack autophagosomes in the cytosol and fail to accumulate autophagic bodies in the vacuole. Although many studies have revealed important details of Atg1 activation and function, a cohesive model for how Atg1 regulates the autophagic machinery is lacking. Our recent findings identified conserved steps of temporal and spatial regulation of Atg1/ULK1 kinase at both the PAS and autophagosomal membranes, suggesting that Atg1 not only promotes autophagy induction, but may also facilitate late stages of autophagosome biogenesis.
PMCID: PMC3552892  PMID: 23108207
Atg1; Atg13; Atg8; LIR; PAS; ULK1; autophagosome; autophagy
6.  No damage of joint cartilage of the lower limbs in an ultra-endurance athlete – an MRI-study 
Osteoarthritis is an increasing burden in an ageing population. Sports, especially when leading to an overstress of joints, is under suspicion to provoke or at least accelerate the genesis of osteoarthritis. We present the radiologic findings of a 49-years old ultra-endurance athlete with 35 years of training and competing, whose joints of the lower limbs were examined using three different types of magnetic resonance imaging, including a microscopic magnetic resonance imaging coil. To date no case report exists where an ultra-endurance athlete was examined such detailed regarding overuse-injuries of his joints.
Case presentation
A 49 years old, white, male ultra-endurance athlete reporting no pain during training and racing and with no significant injuries of the lower limbs in his medical history was investigated regarding signs of chronic damage or overuse injuries of the joints of his lower limbs.
Despite the age of nearly 50 years and a training history of over 35 years, the athlete showed no signs of chronic damage or overuse injuries in the joints of his lower limbs. This leads to the conclusion that extensive sports and training does not compulsory lead to damages of the musculoskeletal system. This is a very important finding for all endurance-athletes as well as for their physicians.
PMCID: PMC3903073  PMID: 24314152
Magnetic resonance imaging; Microscopic magnetic resonance imaging; Over-use injuries; Extreme-endurance; Over-stress; Joint-injuries
7.  Regulating PLK1 dynamics by Cullin3/KLHL22-mediated ubiquitylation 
Cell Cycle  2013;12(16):2528-2529.
PMCID: PMC3865036  PMID: 23907128
8.  Structural basis for a reciprocal regulation between SCF and CSN 
Cell reports  2012;2(3):616-627.
SCF (Skp1-Cul1-Fboxes) E3 ligases are activated by ligation to the ubiquitin-like protein Nedd8, which is reversed by the deneddylating Cop9 Signalosome (CSN). However, CSN also promotes SCF substrate turnover through unknown mechanisms. Through biochemical and electron microscopy analyses, we determined molecular models of CSN complexes with SCFSkp2/Cks1 and SCFFbw7 and found that CSN occludes both SCF functional sites – the catalytic Rbx1-Cul1 C-terminal domain and the substrate receptor. Indeed, CSN binding prevents SCF interactions with E2 enzymes and a ubiquitination substrate, and inhibits SCF-catalyzed ubiquitin chain formation independent of deneddylation. Importantly, CSN prevents neddylation of the bound cullin, unless binding of a ubiquitination substrate triggers SCF dissociation and neddylation. Taken together, the results provide a model for how reciprocal regulation sensitizes CSN to the SCF assembly state, and inhibits a catalytically-competent SCF until a ubiquitination substrate drives its own degradation by displacing CSN, thereby promoting cullin neddylation and substrate ubiquitination.
PMCID: PMC3703508  PMID: 22959436
9.  Fbw7α and Fbw7γ Collaborate To Shuttle Cyclin E1 into the Nucleolus for Multiubiquitylation 
Molecular and Cellular Biology  2013;33(1):85-97.
Cyclin E1, an activator of cyclin-dependent kinase 2 (Cdk2) that promotes replicative functions, is normally expressed periodically within the mammalian cell cycle, peaking at the G1-S-phase transition. This periodicity is achieved by E2F-dependent transcription in late G1 and early S phases and by ubiquitin-mediated proteolysis. The ubiquitin ligase that targets phosphorylated cyclin E is SCFFbw7 (also known as SCFCdc4), a member of the cullin ring ligase (CRL) family. Fbw7, a substrate adaptor subunit, is expressed as three splice-variant isoforms with different subcellular distributions: Fbw7α is nucleoplasmic but excluded from the nucleolus, Fbw7β is cytoplasmic, and Fbw7γ is nucleolar. Degradation of cyclin E in vivo requires SCF complexes containing Fbw7α and Fbw7γ, respectively. In vitro reconstitution showed that the role of SCFFbw7α in cyclin E degradation, rather than ubiquitylation, is to serve as a cofactor of the prolyl cis-trans isomerase Pin1 in the isomerization of a noncanonical proline-proline bond in the cyclin E phosphodegron. This isomerization is required for subsequent binding and ubiquitylation by SCFFbw7γ. Here we show that Pin1-mediated isomerization of the cyclin E phosphodegron and subsequent binding to Fbw7γ drive nucleolar localization of cyclin E, where it is ubiquitylated by SCFFbw7γ prior to its degradation by the proteasome. It is possible that this constitutes a mechanism for rapid inactivation of phosphorylated cyclin E by nucleolar sequestration prior to its multiubiquitylation and degradation.
PMCID: PMC3536299  PMID: 23109421
10.  Cullin-3 and the endocytic system 
Cellular Logistics  2012;2(3):166-168.
Cullin-RING-ligases (CRLs) comprise the largest class of multisubunit E3 ubiquitin ligases, which regulate a broad range of cellular processes. Cullin3 (Cul3) recently emerged as an important regulator of intracellular trafficking, in particular secretion and endosome maturation. Here we summarize and discuss possible functions and substrates of Cul3 in the endocytic system.
PMCID: PMC3498076  PMID: 23739092
endocytosis; ubiquitin; Cullin3; Cul3; EGFR; influenza A virus
11.  The TFIIH subunit Tfb3 regulates cullin neddylation 
Molecular cell  2011;43(3):488-495.
Cullin proteins are scaffolds for the assembly of multi-subunit ubiquitin ligases, which ubiquitylate a large number of proteins involved in widely-varying cellular functions. Multiple mechanisms cooperate to regulate cullin activity, including neddylation of their C-terminal domain. Interestingly, we found that the yeast Cul4-type cullin Rtt101 is not only neddylated but also ubiquitylated, and both modifications promote Rtt101 function in vivo. Surprisingly, proper modification of Rtt101 neither correlated with catalytic activity of the RING-domain of Hrt1 nor did it require the Nedd8 ligase Dcn1. Instead, ubiquitylation of Rtt101 was dependent on the ubiquitin-conjugating enzyme Ubc4, while efficient neddylation involves the RING-domain protein Tfb3, a subunit of the transcription factor TFIIH. Tfb3 also controls Cul3 neddylation and activity in vivo, and physically interacts with Ubc4 and the Nedd8-conjugating enzyme Ubc12 as well as the Hrt1/Rtt101 complex. Together, these results suggest that the conserved RING-domain protein Tfb3 controls activation of a subset of cullins.
PMCID: PMC3186349  PMID: 21816351
12.  Dynamic processes at stress promoters regulate the bimodal expression of HOG response genes 
Osmotic stress triggers the activation of the HOG (high osmolarity glycerol) pathway in Saccharomyces cerevisiae. This signaling cascade culminates in the activation of the MAPK (mitogen-activated protein kinase) Hog1. Quantitative single cell measurements revealed a discrepancy between kinase- and transcriptional activities of Hog1. While kinase activity increases proportionally to stress stimulus, gene expression is inhibited under low stress conditions. Interestingly, a slow stochastic gene activation process is responsible for setting a tunable threshold for gene expression under basal or low stress conditions, which generates a bimodal expression pattern at intermediate stress levels.
PMCID: PMC3306335  PMID: 22446531
MAPK signaling; HOG pathway; cellular stress response; transcription; single cell analysis
13.  Control of Ubp3 ubiquitin protease activity by the Hog1 SAPK modulates transcription upon osmostress 
The EMBO Journal  2011;30(16):3274-3284.
Control of Ubp3 ubiquitin protease activity by the Hog1 SAPK modulates transcription upon osmostress
Here, the Hog1 kinase interacts with and activates the ubiquitin protease Ubp3 in a stress-dependent manner. The phosphorylation of Ubp3 enhances RNA polymerase II occupancy on osmotic stress-responsive genes.
Protein ubiquitylation is a key process in the regulation of many cellular processes. The balance between the activity of ubiquitin ligases and that of proteases controls the level of ubiquitylation. In response to extracellular stimuli, stress-activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK has a key role in reprogramming the gene expression pattern required for cell survival upon osmostress. Here, we show that the Ubp3 ubiquitin protease is a target for the Hog1 SAPK to modulate gene expression. ubp3 mutant cells are defective in expression of osmoresponsive genes. Hog1 interacts with and phosphorylates Ubp3 at serine 695, which is essential to determine the extent of transcriptional activation in response to osmostress. Furthermore, Ubp3 is recruited to osmoresponsive genes to modulate transcriptional initiation as well as elongation. Therefore, Ubp3 activity responds to external stimuli and is required for transcriptional activation upon osmostress.
PMCID: PMC3160652  PMID: 21743437
gene expression; Hog1; osmostress; SAPK; Ubp3
14.  Phosphoproteomic Analysis Reveals Interconnected System-Wide Responses to Perturbations of Kinases and Phosphatases in Yeast 
Science signaling  2010;3(153):rs4.
The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules, and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery, and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.
PMCID: PMC3072779  PMID: 21177495
15.  The Cul3–KLHL21 E3 ubiquitin ligase targets Aurora B to midzone microtubules in anaphase and is required for cytokinesis 
The Journal of Cell Biology  2009;187(6):791-800.
Selective ubiquitination of Aurora B by different Cul3 adaptors targets it at the correct time to the correct place during mitosis.
Cul3 (Cullin3)-based E3 ubiquitin ligases recently emerged as critical regulators of mitosis. In this study, we identify two mammalian BTB (Bric-a-brac–Tramtrack–Broad complex)-Kelch proteins, KLHL21 and KLHL22, that interact with Cul3 and are required for efficient chromosome alignment. Interestingly, KLHL21 but not KLHL22 is necessary for cytokinesis and regulates translocation of the chromosomal passenger complex (CPC) from chromosomes to the spindle midzone in anaphase, similar to the previously described BTB-Kelch proteins KLHL9 and KLHL13. KLHL21 directly binds to Aurora B and mediates ubiquitination of Aurora B in vitro. In contrast to KLHL9 and KLHL13, KLHL21 localizes to midzone microtubules in anaphase and recruits Aurora B and Cul3 to this region. Together, our results suggest that different Cul3 adaptors nonredundantly regulate Aurora B during mitosis, possibly by ubiquitinating different pools of Aurora B at distinct subcellular localizations.
PMCID: PMC2806313  PMID: 19995937
16.  The Clathrin Adaptor Gga2p Is a Phosphatidylinositol 4-phosphate Effector at the Golgi Exit 
Molecular Biology of the Cell  2008;19(5):1991-2002.
Phosphatidylinositol 4-phosphate (PI(4)P) is a key regulator of membrane transport required for the formation of transport carriers from the trans-Golgi network (TGN). The molecular mechanisms of PI(4)P signaling in this process are still poorly understood. In a search for PI(4)P effector molecules, we performed a screen for synthetic lethals in a background of reduced PI(4)P and found the gene GGA2. Our analysis uncovered a PI(4)P-dependent recruitment of the clathrin adaptor Gga2p to the TGN during Golgi-to-endosome trafficking. Gga2p recruitment to liposomes is stimulated both by PI(4)P and the small GTPase Arf1p in its active conformation, implicating these two molecules in the recruitment of Gga2p to the TGN, which ultimately controls the formation of clathrin-coated vesicles. PI(4)P binding occurs through a phosphoinositide-binding signature within the N-terminal VHS domain of Gga2p resembling a motif found in other clathrin interacting proteins. These data provide an explanation for the TGN-specific membrane recruitment of Gga2p.
PMCID: PMC2366843  PMID: 18287542
17.  Regulation of Rtt107 Recruitment to Stalled DNA Replication Forks by the Cullin Rtt101 and the Rtt109 Acetyltransferase 
Molecular Biology of the Cell  2008;19(1):171-180.
RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint kinase Mec1, and it forms complexes with DNA repair enzymes, including the nuclease subunit Slx4, but the role of Rtt107 in the DNA damage response remains unclear. We find that Rtt107 interacts with chromatin when cells are treated with compounds that cause replication forks to arrest. This damage-dependent chromatin binding requires the acetyltransferase Rtt109, but it does not require acetylation of the known Rtt109 target, histone H3-K56. Chromatin binding of Rtt107 also requires the cullin Rtt101, which seems to play a direct role in Rtt107 recruitment, because the two proteins are found in complex with each other. Finally, we provide evidence that Rtt107 is bound at or near stalled replication forks in vivo. Together, these results indicate that Rtt109, Rtt101, and Rtt107, which genetic evidence suggests are functionally related, form a DNA damage response pathway that recruits Rtt107 complexes to damaged or stalled replication forks.
PMCID: PMC2174167  PMID: 17978089
18.  Annotating novel genes by integrating synthetic lethals and genomic information 
Large scale screening for synthetic lethality serves as a common tool in yeast genetics to systematically search for genes that play a role in specific biological processes. Often the amounts of data resulting from a single large scale screen far exceed the capacities of experimental characterization of every identified target. Thus, there is need for computational tools that select promising candidate genes in order to reduce the number of follow-up experiments to a manageable size.
We analyze synthetic lethality data for arp1 and jnm1, two spindle migration genes, in order to identify novel members in this process. To this end, we use an unsupervised statistical method that integrates additional information from biological data sources, such as gene expression, phenotypic profiling, RNA degradation and sequence similarity. Different from existing methods that require large amounts of synthetic lethal data, our method merely relies on synthetic lethality information from two single screens. Using a Multivariate Gaussian Mixture Model, we determine the best subset of features that assign the target genes to two groups. The approach identifies a small group of genes as candidates involved in spindle migration. Experimental testing confirms the majority of our candidates and we present she1 (YBL031W) as a novel gene involved in spindle migration. We applied the statistical methodology also to TOR2 signaling as another example.
We demonstrate the general use of Multivariate Gaussian Mixture Modeling for selecting candidate genes for experimental characterization from synthetic lethality data sets. For the given example, integration of different data sources contributes to the identification of genetic interaction partners of arp1 and jnm1 that play a role in the same biological process.
PMCID: PMC2258006  PMID: 18194531
19.  Saccharomyces cerevisiae Ebs1p is a putative ortholog of human Smg7 and promotes nonsense-mediated mRNA decay 
Nucleic Acids Research  2007;35(22):7688-7697.
The Smg proteins Smg5, Smg6 and Smg7 are involved in nonsense-mediated RNA decay (NMD) in metazoans, but no orthologs have been found in the budding yeast Saccharomyces cerevisiae. Sequence alignments reveal that yeast Ebs1p is similar in structure to the human Smg5-7, with highest homology to Smg7. We demonstrate here that Ebs1p is involved in NMD and behaves similarly to human Smg proteins. Indeed, both loss and overexpression of Ebs1p results in stabilization of NMD targets. However, Ebs1-loss in yeast or Smg7-depletion in human cells only partially disrupts NMD and in the latter, Smg7-depletion is partially compensated for by Smg6. Ebs1p physically interacts with the NMD helicase Upf1p and overexpressed Ebs1p leads to recruitment of Upf1p into cytoplasmic P-bodies. Furthermore, Ebs1p localizes to P-bodies upon glucose starvation along with Upf1p. Overall our findings suggest that NMD is more conserved in evolution than previously thought, and that at least one of the Smg5-7 proteins is conserved in budding yeast.
PMCID: PMC2190716  PMID: 17984081
20.  CIF-1, a Shared Subunit of the COP9/Signalosome and Eukaryotic Initiation Factor 3 Complexes, Regulates MEL-26 Levels in the Caenorhabditis elegans Embryo▿  
Molecular and Cellular Biology  2007;27(12):4526-4540.
The COP9/signalosome (CSN) is an evolutionarily conserved macromolecular complex that regulates the cullin-RING ligase (CRL) class of E3 ubiquitin ligases, primarily by removing the ubiquitin-like protein Nedd8 from the cullin subunit. In the Caenorhabditis elegans embryo, the CSN controls the degradation of the microtubule-severing protein MEI-1 through CUL-3 deneddylation. However, the molecular mechanisms of CSN function and its subunit composition remain to be elucidated. Here, using a proteomic approach, we have characterized the CSN and CUL-3 complexes from C. elegans embryos. We show that the CSN physically interacts with the CUL-3-based CRL and regulates its activity by counteracting the autocatalytic instability of the substrate-specific adaptor MEL-26. Importantly, we identified the uncharacterized protein K08F11.3/CIF-1 (for CSN-eukaryotic initiation factor 3 [eIF3]) as a stoichiometric and functionally important subunit of the CSN complex. CIF-1 appears to be the only ortholog of Csn7 encoded by the C. elegans genome, but it also exhibits extensive sequence similarity to eIF3m family members, which are required for the initiation of protein translation. Indeed, CIF-1 binds eIF-3.F and inactivation of cif-1 impairs translation in vivo. Taken together, our results indicate that CIF-1 is a shared subunit of the CSN and eIF3 complexes and may therefore link protein translation and degradation.
PMCID: PMC1900047  PMID: 17403899
21.  Selective Requirement for SAGA in Hog1-Mediated Gene Expression Depending on the Severity of the External Osmostress Conditions▿  
Molecular and Cellular Biology  2007;27(11):3900-3910.
Regulation of gene expression by the Hog1 stress-activated protein kinase is essential for proper cell adaptation to osmostress. Hog1 coordinates an extensive transcriptional program through the modulation of transcription. To identify systematically novel components of the transcriptional machinery required for osmostress-mediated gene expression, we performed an exhaustive genome-wide genetic screening, searching for mutations that render cells osmosensitive at high osmolarity and that are associated with reduced expression of osmoresponsive genes. The SAGA and Mediator complexes were identified as putative novel regulators of osmostress-mediated transcription. Interestingly, whereas Mediator is essential for osmostress gene expression, the requirement for SAGA is different depending on the strength of the extracellular osmotic conditions. At mild osmolarity, SAGA mutants show only very slight defects on RNA polymerase II (Pol II) recruitment and gene expression, whereas at severe osmotic conditions, SAGA mutants show completely impaired RNA Pol II recruitment and transcription of osmoresponsive genes. Thus, our results define an essential role for Mediator in osmostress gene expression and a selective role for SAGA under severe osmostress. Our results indicate that the requirement for a transcriptional complex to regulate a promoter might be determined by the strength of the stimuli perceived by the cell through the regulation of interactions between transcriptional complexes.
PMCID: PMC1900016  PMID: 17403898
22.  The GTPase-Activating Enzyme Gyp1p Is Required for Recycling of Internalized Membrane Material by Inactivation of the Rab/Ypt GTPase Ypt1p 
Molecular and Cellular Biology  2004;24(9):3815-3826.
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 gyp1Δ 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.
PMCID: PMC387746  PMID: 15082776
23.  Skp1p and the F-Box Protein Rcy1p Form a Non-SCF Complex Involved in Recycling of the SNARE Snc1p in Yeast 
Molecular and Cellular Biology  2001;21(9):3105-3117.
Skp1p–cullin–F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex. Here we show that the F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p in Saccharomyces cerevisiae. Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton. Rcy1p interacted with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impaired recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p did not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p required neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p was the only major partner that copurified with Rcy1p. Our results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins.
PMCID: PMC86938  PMID: 11287615
24.  Gic2p May Link Activated Cdc42p to Components Involved in Actin Polarization, Including Bni1p and Bud6p (Aip3p) 
Molecular and Cellular Biology  2000;20(17):6244-6258.
Gic2p is a Cdc42p effector which functions during cytoskeletal organization at bud emergence and in response to pheromones, but it is not understood how Gic2p interacts with the actin cytoskeleton. Here we show that Gic2p displayed multiple genetic interactions with Bni1p, Bud6p (Aip3p), and Spa2p, suggesting that Gic2p may regulate their function in vivo. In support of this idea, Gic2p cofractionated with Bud6p and Spa2p and interacted with Bud6p by coimmunoprecipitation and two-hybrid analysis. Importantly, localization of Bni1p and Bud6p to the incipient bud site was dependent on active Cdc42p and the Gic proteins but did not require an intact actin cytoskeleton. We identified a conserved domain in Gic2p which was necessary for its polarization function but dispensable for binding to Cdc42p-GTP and its localization to the site of polarization. Expression of a mutant Gic2p harboring a single-amino-acid substitution in this domain (Gic2pW23A) interfered with polarized growth in a dominant-negative manner and prevented recruitment of Bni1p and Bud6p to the incipient bud site. We propose that at bud emergence, Gic2p functions as an adaptor which may link activated Cdc42p to components involved in actin organization and polarized growth, including Bni1p, Spa2p, and Bud6p.
PMCID: PMC86099  PMID: 10938101
25.  Coronin Promotes the Rapid Assembly and Cross-linking of Actin Filaments and May Link the Actin and Microtubule Cytoskeletons in Yeast  
The Journal of Cell Biology  1999;144(1):83-98.
Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 × 10−9 M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Δ deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Δ cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.
PMCID: PMC2148128  PMID: 9885246
actin; microtubule; coronin; cytoskeleton; yeast

Results 1-25 (25)