Global analysis of protein localization in budding yeast

Huh, Won-Ki; Falvo, James V.; Gerke, Luke C.; Carroll, Adam S.; Howson, Russell W.; Weissman, Jonathan S.; O’Shea, Erin K.

doi:10.1038/nature02026

Cited by 3,917 publications

(4,096 citation statements)

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“…Our vectors can integrate into commonly used auxotrophic yeast strains, including the designer deletion strains (Brachmann et al, 1998), making them compatible with the diverse strain collections (Winzeler et al, 1999;Huh et al, 2003;Ghaemmaghami et al, 2003;Mnaimneh et al, 2004). Efficient integration into the various alleles (carrying point mutations, insertions, partial or complete deletions) is facilitated by using marker genes on the plasmids without any overlap with the host genome (Wach et al, 1997;Goldstein et al, 1999;Gueldener et al, 2002 For the extrachromosomal maintenance of genes at low or high copy number, the series contains centromeric and episomal shuttle vectors, respectively.…”

Section: Discussionmentioning

confidence: 99%

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Gnügge

Liphardt

Rudolf

2016

Yeast

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Shuttle vectors allow for an efficient transfer of recombinant DNA into yeast cells and are widely used in fundamental research and biotechnology. While available shuttle vectors are applicable in many experimental settings, their use in quantitative biology is hampered by insufficient copy number control. Moreover, they often have practical constraints, such as limited modularity and few unique restriction sites. We constructed the pRG shuttle vector series, consisting of single-and multi-copy integrative, centromeric and episomal plasmids with marker genes for the selection in all commonly used auxotrophic yeast strains. The vectors feature a modular design and a large number of unique restriction sites, enabling an efficient exchange of every vector part and expansion of the series. Integration into the host genome is achieved using a double-crossover recombination mechanism, resulting in stable single-and multi-copy modifications. As centromeric and episomal plasmids give rise to a heterogeneous cell population, an analysis of their copy number distribution and loss behaviour was performed. Overall, the shuttle vector series supports the efficient cloning of genes and their maintenance in yeast cells with improved copy number control.

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Section: Discussionmentioning

confidence: 99%

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Gnügge

Liphardt

Rudolf

2016

Yeast

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“…Deubiquitinating enzymes in the cyto- and nucleoplasm provide an additional level on the plasticity on the repertoire of proteasomal substrates ( Sahtoe & Sixma, 2015). Intriguingly, GFP-labelled ubiquitin and the E1, named UBA1, is primarily nuclear in growing yeast and mammalian cells suggesting that ubiquitin-dependent proteolysis mainly occurs in the nucleus ( Huh et al , 2003; Salomons et al , 2010; Sugaya et al , 2014; Sugaya et al , 2015). …”

Section: Discussion/analysis Of the Literaturementioning

confidence: 99%

Intracellular Dynamics of the Ubiquitin-Proteasome-System

Chowdhury

Enenkel

2015

F1000Res

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The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum (ER) membranes. In prolonged quiescence, proteasome granules drop off the NE / ER membranes and migrate as stable organelles throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus.Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm.Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells which comprise the majority of our body’s cells.

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“…truncatula hairy roots, bombarded onion slices and transformed BY4742 yeast cells (with an integrated RFP tagged Sec13 protein for ER and Golgi marking) 48,49 were analyzed by confocal microscopy with the FV10 ASW Olympus Confocal with a water immersion 63× objective.…”

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confidence: 99%

The protein quality control system manages plant defence compound synthesis

Pollier

Moses

González-Guzmán

et al. 2013

Nature

126

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Jasmonates (JAs) are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of multiple plant processes, encompassing development, growth and defense. Across the plant kingdom, JAs act as elicitors of the production of bioactive secondary metabolites that serve in the defense against attackers 1-3 . Knowledge on the conserved JA perception and early signaling machineries is increasing 3-6 but the downstream mechanisms that regulate defense metabolism remain largely unknown.Here we show that in the model legume Medicago truncatula JA recruits the endoplasmic reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols 7-9 . An ERAD-type RING membrane-anchor (RMA) E3 ubiquitin (Ub)-ligase is co-expressed with saponin synthesis enzymes to control 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thereby unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that employs distinct E3 Ub-ligases, of the HMGR Degradation 1 (HRD1)-type, to direct destruction of HMGR 10-13 . Hence, the general principles for management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues. 3To identify regulators of plant triterpene synthesis, we monitored the transcriptome of suspension-cultured M. truncatula cells, known to accumulate saponins following elicitation with JAs 14 . The expression of 8,462 transcripts was visualized by cDNA-AFLP transcript profiling and 282 Methyl JA (MeJA)-responsive tags were identified. The comparable MeJAinduced expression pattern of the genes encoding HMGR, squalene synthase, squalene epoxidase, β-amyrin synthase (BAS) and CYP93E2, enzymes catalyzing steps in triterpene saponin biosynthesis 7-9 , indicated co-regulation ( Fig. 1a and Extended Data Fig. 1-2). Several genes corresponding to potential regulatory factors had maximal transcriptional upregulation prior to or concurrent with that of the triterpene saponin genes, including a MYC-like bHLH protein and the JAZ repressor proteins, known elements of the core JA signaling module 4-6 , as well as a gene (MT067) corresponding to an RMA-like E3 Ub-ligase 10 , denominated MAKIBISHI1 (MKB1) (Extended Data Fig. 3). The early MeJA response of MKB1 was confirmed in the M. truncatula Gene Expression Atlas (MtGEA; http://bioinfo.noble.org/geneatlas/) 15 (Fig. 1b).To assess MKB1 function, we generated transgenic M. truncatula hairy roots in which MKB1 was overexpressed (MKB1 OE roots) or silenced (MKB1 KD roots) (Extended Data Fig. 4a). MKB1 KD roots showed a striking phenotype, in particular when transferred to liquid medium, which caused 'dissociation' of the MKB1 KD roots into 'c...

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Global analysis of protein localization in budding yeast

Cited by 3,917 publications

References 47 publications

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Intracellular Dynamics of the Ubiquitin-Proteasome-System

The protein quality control system manages plant defence compound synthesis

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