Atencio D, Barnes C, Duncan TM, Willis IM, Hanes SD
G3 (Bethesda). Genetics Society of America; 2014 Mar 1;4(3):523–37
Yeast/SGA
The Ess1 prolyl isomerase from Saccharomyces cerevisiae and its human ortholog, Pin1, play critical roles in transcription by regulating RNA polymerase II. In human cells, Pin1 also regulates a variety of signaling proteins, and Pin1 misexpression is linked to several human diseases. To gain insight into Ess1/Pin1 function, we carried out a synthetic genetic array screen to identify novel targets of Ess1 in yeast. We identified potential targets of Ess1 in transcription, stress, and cell-cycle pathways. We focused on the cell-cycle regulators Swi6 and Whi5, both of which show highly regulated nucleocytoplasmic shuttling during the cell cycle. Surprisingly, Ess1 did not control their transcription but instead was necessary for theirnuclear localization. Ess1 associated with Swi6 and Whi5 in vivo and bound directly to peptides corresponding to their nuclear localizationsequences in vitro. Binding by Ess1 was significant only if the Swi6 and Whi5 peptides were phosphorylated at Ser-Pro motifs, the target sites of cyclin-dependent kinases. On the basis of these results, we propose a model in which Ess1 induces a conformational switch (cis-trans isomerization) at phospho-Ser-Pro sites within the nuclear targeting sequences of Swi6 and Whi5. This switch would promote nuclear entry and/or retention during late M and G1 phases and might work by stimulating dephosphorylation at these sites by the Cdc14 phosphatase. This is the first study to identify targets of Ess1 in yeast other than RNA polymerase II.
Vieira NM, Naslavsky MS, Licinio L, Kok F, Schlesinger D, Vainzof M, Sanchez N, Kitajima JP, Gal L, Cavaçana N, Serafini PR, Chuartzman S, Vasquez C, Mimbacas A, Nigro V, Pavanello RC, Schuldiner M, Kunkel LM, Zatz M
Hum Mol Genet. 2014 Aug 1;23(15):4103-10
Yeast/SGA
Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of genetically determined muscle disorders with a primary or predominant involvement of the pelvic or shoulder girdle musculature. More than 20 genes with autosomal recessive (LGMD2A to LGMD2Q) and autosomal dominant inheritance (LGMD1A to LGMD1H) have been mapped/identified to date. Mutations are known for six among the eight mapped autosomal dominant forms: LGMD1A (myotilin), LGMD1B (lamin A/C), LGMD1C (caveolin-3), LGMD1D (desmin), LGMD1E (DNAJB6), and more recently for LGMD1F (transportin-3). Our group previously mapped the LGMD1G gene at 4q21 in a Caucasian-Brazilian family. We now mapped a Uruguayan family with patients displaying a similar LGMD1G phenotype at the same locus. Whole genome sequencing identified, in both families, mutations in the HNRPDL gene. HNRPDL is a heterogeneous ribonucleoprotein family member, which participates in mRNA biogenesis and metabolism. Functional studies performed in S. cerevisiae showed that the loss of HRP1 (yeast orthologue) had pronounced effects on both protein levels and cell localizations, and yeast proteome revealed dramatic reorganization of proteins involved in RNA-processing pathways. In vivo analysis showed that hnrpdl is important for muscle development in zebrafish, causing a myopathic phenotype when knocked down. The present study presents a novel association between a muscular disorder and a RNA-related gene and reinforces the importance of RNA binding/processing proteins in muscle development and muscle disease. Understanding the role of these proteins in muscle might open new therapeutic approaches for musculardystrophies.
Ast T, Aviram N, Chuartzman SG, Schuldiner M
J Cell Sci. 2014 Jul 15;127(Pt 14):3017-2
Yeast/SGA
The endoplasmic reticulum (ER) identifies and disposes of misfolded secretory pathway proteins through the actions of ER-associated degradation(ERAD) pathways. It is becoming evident that a substantial fraction of the secretome transiently resides in the cytosol before translocating into the ER, both in yeast and in higher eukaryotes. To uncover factors that monitor this transient cytosolic protein pool, we carried out a genetic screen in Saccharomyces cerevisiae. Our findings highlighted a pre-insertional degradation mechanism at the cytosolic leaflet of the ER, which we termprERAD. prERAD relies on the concurrent action of the ER-localized ubiquitylation and deubiquitylation machineries Doa10 and Ubp1. By recognizing C-terminal hydrophobic motifs, prERAD tags for degradation pre-inserted proteins that have remained on the cytosolic leaflet of the ER for too long. Our discoveries delineate a new cellular safeguard, which ensures that every stage of secretory pathway protein biogenesis is scrutinized and regulated.
Cohen Y, Klug YA, Dimitrov L, Erez Z, Chuartzman SG, Elinger D, Yofe I, Soliman K, Gärtner J, Thoms S, Schekman R, Elbaz-Alon Y, Zalckvar E, Schuldiner M
Mol BioSyst. The Royal Society of Chemistry; 2014 Jun 3;10(7):1742–8
Yeast/SGA
Peroxisomes are ubiquitous and dynamic organelles that house many important pathways of cellular metabolism. In recent years it has been demonstrated that mitochondria are tightly connected with peroxisomes and are defective in several peroxisomal diseases. Indeed, these two organelles share metabolic routes as well as resident proteins and, at least in mammals, are connected via a vesicular transport pathway. However the exact extent of cross-talk between peroxisomes and mitochondria remains unclear. Here we used a combination of high throughput genetic manipulations of yeast libraries alongside high content screens to systematically unravel proteins that affect the transport of peroxisomal proteins and peroxisome biogenesis. Follow up work on the effector proteins that were identified revealed that peroxisomes are not randomly distributed in cells but are rather localized to specific mitochondrial subdomains such as mitochondria-ER junctions and sites of acetyl-CoA synthesis. Our approach highlights the intricate geography of the cell and suggests an additional layer of organization as a possible way to enable efficient metabolism. Our findings pave the way for further studying the machinery aligning mitochondria and peroxisomes, the role of the juxtaposition, as well as its regulation during various metabolic conditions. More broadly, the approaches used here can be easily applied to study any organelle of choice, facilitating the discovery of new aspects in cell biology.
Figley MD, Bieri G, Kolaitis R-M, Taylor JP, Gitler AD
J Neurosci. 2014 Jun 11;34(24):8083-97
Yeast/SGA
Mutations in the PFN1 gene encoding profilin 1 are a rare cause of familial amyotrophic lateral sclerosis (ALS). Profilin 1 is a well studied actin-binding protein but how PFN1 mutations cause ALS is unknown. The budding yeast, Saccharomyces cerevisiae, has one PFN1 ortholog. We expressed the ALS-linked profilin 1 mutant proteins in yeast, demonstrating a loss of protein stability and failure to restore growth to profilin mutant cells, without exhibiting gain-of-function toxicity. This model provides for simple and rapid screening of novel ALS-linked PFN1 variants. To gain insight into potential novel roles for profilin 1, we performed an unbiased, genome-wide synthetic lethal screen with yeast cells lacking profilin(pfy1Δ). Unexpectedly, deletion of several stress granule and processing body genes, including pbp1Δ, were found to be synthetic lethal with pfy1Δ.Mutations in ATXN2, the human ortholog of PBP1, are a known ALS genetic risk factor and ataxin 2 is a stress granule component in mammalian cells. Given this genetic interaction and recent evidence linking stress granule dynamics to ALS pathogenesis, we hypothesized that profilin 1 might also associate with stress granules. Here we report that profilin 1 and related protein profilin 2 are novel stress granule-associated proteins in mouse primary cortical neurons and in human cell lines and that ALS-linked mutations in profilin 1 alter stress granule dynamics, providing further evidence for the potential role of stress granules in ALS pathogenesis.
Kayatekin C, Matlack KES, Hesse WR, Guan Y, Chakrabortee S, Russ J, Wanker EE, Shah JV, Lindquist S
Proc Natl Acad Sci USA. National Acad Sciences; 2014 Aug 19;111(33):12085–90
Yeast/SGA
Expansions of preexisting polyglutamine (polyQ) tracts in at least nine different proteins cause devastating neurodegenerative diseases. There are many unique features to these pathologies, but there must also be unifying mechanisms underlying polyQ toxicity. Using a polyQ-expanded fragment of huntingtin exon-1 (Htt103Q), the causal protein in Huntington disease, we and others have created tractable models for investigating polyQ toxicityin yeast cells. These models recapitulate key pathological features of human diseases and provide access to an unrivalled genetic toolbox. To identify toxicity modifiers, we performed an unbiased overexpression screen of virtually every protein encoded by the yeast genome. Surprisingly, there was no overlap between our modifiers and those from a conceptually identical screen reported recently, a discrepancy we attribute to an artifact of their overexpression plasmid. The suppressors of Htt103Q toxicity recovered in our screen were strongly enriched for glutamine- and asparagine-rich prion-like proteins. Separated from the rest of the protein, the prion-like sequences of these proteins were themselves potent suppressors of polyQ-expanded huntingtin exon-1 toxicity, in both yeast and human cells. Replacing the glutamines in these sequences with asparagines abolished suppression and converted them to enhancers of toxicity. Replacing asparagines with glutamines created stronger suppressors. The suppressors (but not the enhancers) coaggregated with Htt103Q, forming large foci at the insoluble protein deposit in which proteins were highly immobile. Cells possessing foci had fewer (if any) small diffusible oligomers of Htt103Q. Until such foci were lost, cells were protected from death. We discuss the therapeutic implications of these findings.
Dhungel N, Eleuteri S, Li L-B, Kramer NJ, Chartron JW, Spencer B, Kosberg K, Fields JA, Stafa K, Adame A, Lashuel H, Frydman J, Shen K, Masliah E, Gitler AD
Neuron. 2015 Jan;85(1):76–87
Yeast/SGA
Parkinson's disease (PD) is a common neurodegenerative disorder. Functional interactions between some PD genes, like PINK1 and parkin, have been identified, but whether other ones interact remains elusive. Here we report an unexpected genetic interaction between two PD genes, VPS35and EIF4G1. We provide evidence that EIF4G1 upregulation causes defects associated with protein misfolding. Expression of a sortilin protein rescues these defects, downstream of VPS35, suggesting a potential role for sortilins in PD. We also show interactions between VPS35, EIF4G1, and α-synuclein, a protein with a key role in PD. We extend our findings from yeast to an animal model and show that these interactions are conserved in neurons and in transgenic mice. Our studies reveal unexpected genetic and functional interactions between two seemingly unrelated PD genes and functionally connect them to α-synuclein pathobiology in yeast, worms, and mouse. Finally, we provide a resource of candidate PDgenes for future interrogation.
Sanchez-Casalongue ME, Lee J, Diamond A, Shuldiner S, Moir RD, Willis IM
J Biol Chem. American Society for Biochemistry and Molecular Biology; 2015 Jan 28;:jbc.M114.626523
Yeast/SGA
Transcriptional regulation of ribosome and tRNA synthesis plays a central role in determining protein synthetic capacity and is tightly controlled in response to nutrient availability and cellular stress. In Saccharomyces cerevisiae, the regulation of ribosome and tRNA synthesis was recently shown to involve the Cdc-like kinase Kns1 and the GSK-3 kinase Mck1. In this study, we explored additional roles for these conserved kinases in processes connected to the Target of Rapamycin Complex 1 (TORC1). We conducted a synthetic chemical-genetic screen in a kns1∆ mck1∆ strain and identified many novel rapamycin-hypersensitivity genes. Gene ontology analysis showed enrichment for TORC1-regulated processes (vesicle-mediated transport, autophagy and regulation of cell size) and identified new connections to protein complexes including the protein kinase CK2. CK2is considered to be a constitutively active kinase and in budding yeast, the holoenzyme comprises two regulatory subunits, Ckb1 and Ckb2, and two catalytic subunits, Cka1 and Cka2. We show that Ckb1 is differentially phosphorylated in vivo and that Kns1 mediates this phosphorylation when nutrients are limiting and under all tested stress conditions. We determined that the phosphorylation of Ckb1 does not detectably affect the stability of the CK2 holoenzyme but correlates with the reduced occupancy of Ckb1 on tRNA genes after rapamycin treatment. Thus, the differentialoccupancy of tRNA genes by CK2 is likely to modulate its activation of RNA polymerase III transcription. Our data suggest that TORC1, via its effector kinase Kns1, may regulate the association of CK2 with some of its substrates by phosphorylating Ckb1.
Fournier ML, Paulson A, Pavelka N, Mosley AL, Gaudenz K, Bradford WD, Glynn E, Li H, Sardiu ME, Fleharty B, Seidel C, Florens L, Washburn MP
Mol Cell Proteomics. 2010 Feb;9(2):271–84
Yeast/Chemical Genetics
To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with proteinexpression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expressionmight be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of therapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence ofrapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Deltaggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression under TOR control.