A novel yeast expression system for the overproduction of quality‐controlled membrane proteins

Griffith, Douglas A.; Delipala, Christina; Leadsham, Jane E.; Jarvis, Simon M.; Oesterhelt, Dieter

doi:10.1016/s0014-5793(03)00952-9

Cited by 53 publications

(39 citation statements)

References 44 publications

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“…To further ascertain the role of UPR in hypoxia tolerance, we decided to examine the effect of knocking out key components of the UPR signaling pathway (16,39,78,85) on the hypoxia tolerance of the mutants with enhanced hypoxia tolerance. Gcn4 was shown to be essential for UPR and is required for the induction of a majority of UPR target genes (85).…”

Section: Resultsmentioning

confidence: 99%

Deletion of a subgroup of ribosome-related genes minimizes hypoxia-induced changes and confers hypoxia tolerance

Shah

Cadinu

Henke

et al. 2011

Physiological Genomics

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Deletion of a subgroup of ribosome-related genes minimizes hypoxia-induced changes and confers hypoxia tolerance. Physiol Genomics 43: 855-872, 2011. First published May 17, 2011 doi:10.1152/physiolgenomics.00232.2010.-Hypoxia is a widely occurring condition experienced by diverse organisms under numerous physiological and disease conditions. To probe the molecular mechanisms underlying hypoxia responses and tolerance, we performed a genome-wide screen to identify mutants with enhanced hypoxia tolerance in the model eukaryote, the yeast Saccharomyces cerevisiae.Yeast provides an excellent model for genomic and proteomic studies of hypoxia. We identified five genes whose deletion significantly enhanced hypoxia tolerance. They are RAI1, NSR1, BUD21, RPL20A, and RSM22, all of which encode functions involved in ribosome biogenesis. Further analysis of the deletion mutants showed that they minimized hypoxia-induced changes in polyribosome profiles and protein synthesis. Strikingly, proteomic analysis by using the iTRAQ profiling technology showed that a substantially fewer number of proteins were changed in response to hypoxia in the deletion mutants, compared with the parent strain. Computational analysis of the iTRAQ data indicated that the activities of a group of regulators were regulated by hypoxia in the wild-type parent cells, but such regulation appeared to be diminished in the deletion strains. These results show that the deletion of one of the genes involved in ribosome biogenesis leads to the reversal of hypoxia-induced changes in gene expression and related regulators. They suggest that modifying ribosomal function is an effective mechanism to minimize hypoxia-induced specific protein changes and to confer hypoxia tolerance. These results may have broad implications in understanding hypoxia responses and tolerance in diverse eukaryotes ranging from yeast to humans.genome-wide screen; proteomic analysis; iTRAQ; regulatory network; stress response LIVING ORGANISMS RANGING FROM aquatic organisms to plants and humans can experience episodes of low oxygen availability, namely, hypoxia. Hypoxia can occur in plant roots when a field is flooded, in yeast during fermentation, or in humans at high altitudes or as a result of a heart attack or stroke. Because oxygen is critical for the survival and development of many living organisms, particularly eukaryotes, living organisms ranging from yeast to humans have developed sophisticated mechanisms to respond and adapt to the changes in oxygen levels in the environment (12, 127). In the wilderness, several species, including fossorial mammals, diving animals, and birds living at high altitudes, have acquired hypoxia tolerance, which enables them to survive under conditions of limited oxygen supply (9, 84, 92). In humans, hypoxia is associated with an array of pathological conditions, such as cancer and stroke. Mechanisms of oxygen sensing and regulation have been implicated in a wide array of physiological and pathological processes (35,99,127). Thus, studying hypoxia ...

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Section: Resultsmentioning

confidence: 99%

Deletion of a subgroup of ribosome-related genes minimizes hypoxia-induced changes and confers hypoxia tolerance

Shah

Cadinu

Henke

et al. 2011

Physiological Genomics

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“…The fact that D1 is an integral membrane protein limits the choice of expression systems to eukaryotic cells, for instance yeast or insect cells, since expression in bacteria of D1 protein results in the production of inactive D1 that accumulates in inclusion bodies (G G J M Kuiper, W Klootwijk & T J Visser, unpublished observations). A heterologous expression system that combines several advantages of both prokaryotic and eukaryotic expression systems are yeasts such as Saccharomyces cerevisiae and Pichia pastoris (Guengerich et al 1991, Bill 2001, Griffith et al 2003. Yeast cells can perform proper post-translational modifications, and contain identical secretory pathways as higher eukaryotic cells.…”

Section: N-bromoacetyl-[mentioning

confidence: 99%

“…In this system proteins are expressed under the control of the GAL1 promoter, allowing propagation of transformed cells in medium with a noninducing carbon source (glucose) and subsequent induction by the addition of galactose (Guengerich et al 1991). An important feature of this system is the possibility to use different galactose induction times in order to avoid saturation of the cell's protein folding capacity (Griffith et al 2003).…”

Section: Kinetic Analysis Of D1 Activity In Yeast Subcellular Fractionsmentioning

confidence: 99%

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Kuiper

Klootwijk²,

Visser³

2005

Journal of Molecular Endocrinology

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The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone thyroxine (T4) by the hepatic selenoenzyme type I iodothyronine deiodinase (D1), i.e. by outer ring deiodination (ORD) to the active hormone triiodothyronine (T3) or by inner ring deiodination (IRD) to the inactive metabolite reverse T3 (rT3). Since D1 is a membrane-bound protein with an N-terminal membrane-spanning domain, the enzyme is very difficult to purify in an active state. This study was undertaken in order to develop a heterologous (over)-expression system that would eventually allow the production of large amounts of purified active D1 protein. We have expressed a mutant rat D1 protein, in which the selenocysteine residue in the core catalytic center was replaced by cysteine (D1 Cys) in yeast cells (Saccharomyces cerevisiae). After yeast cell fractionation, kinetic analysis was performed with dithiothreitol as reducing cofactor. ORD activity was associated with membrane fractions, while no activity could be detected in the cytosolic fraction. The D1 Cys protein displayed a tenfold increase in K m (2 µM) for rT3 as compared with native D1 protein in rat liver microsomes. The D1 protein content is about 65 pmol/mg microsomal protein, as compared with about 3 pmol/mg in rat liver microsomal fraction. SDS-PAGE analysis of N-bromoacetyl-[125 I]T3 affinity-labeled D1 protein showed several labeled protein isoforms with apparent molecular masses between 27 and 32 kDa. Immunoblot analysis with a specific D1 antiserum confirmed the observed D1 protein heterogeneity. Site-directed mutagenesis of several potential N-linked glycosylation sites, phosphorylation sites and a unique myristoylation site established that D1 heterogeneity is not caused by N-linked glycosylation, but probably by a combination of O-linked glycosylation and phosphorylation. Deletion of the endoplasmic reticulum (ER)-signal sequence and the membrane-spanning domain (amino acid residue 2-35), did not result in the production of a soluble D1 enzyme. Although this mutated D1 protein is inactive, the fact that it is still membrane bound indicates the existence of additional membrane attachment site(s) or membrane-spanning domains. Overall, our studies indicate that yeast cells provide a useful system for the expression of relatively high levels of D1 protein which could be used for further structure-function analysis.

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“…The NuoL N fusion proteins were inserted into intracellular membranes without a speciWc leader sequence, as reported for other membrane proteins which were heterologously produced in S. cerevisiae (GriYth et al 2003). We studied the orientation of ProtA-NuoL N in ER vesicles by comparing the accessibility of the protein A epitope before and after solubilization of the vesicles with Triton X-100.…”

Section: Resultsmentioning

confidence: 99%

Transport of Na+ and K+ by an antiporter-related subunit from the Escherichia coli NADH dehydrogenase I produced in Saccharomyces cerevisiae

et al. 2007

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The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na + rather than H + . To elucidate the mechanism of Na + transport, the C-terminally truncated NuoL subunit (NuoL N ) which is related to Na + /H + antiporters was expressed as a protein A fusion protein (ProtANuoL N ) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as conWrmed by diVerential centrifugation and Western analysis. Membrane vesicles containing ProtA-NuoL N catalyzed the uptake of Na + and K + at rates which were signiWcantly higher than uptake by the control vesicles under identical conditions, demonstrating that ProtA-NuoL N translocated Na + and K + independently from other complex I subunits. Na + transport by ProtA-NuoL N was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which speciWcally reacts with Na + /H + antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex is discussed.

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A novel yeast expression system for the overproduction of quality‐controlled membrane proteins

Cited by 53 publications

References 44 publications

Deletion of a subgroup of ribosome-related genes minimizes hypoxia-induced changes and confers hypoxia tolerance

Deletion of a subgroup of ribosome-related genes minimizes hypoxia-induced changes and confers hypoxia tolerance

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Transport of Na+ and K+ by an antiporter-related subunit from the Escherichia coli NADH dehydrogenase I produced in Saccharomyces cerevisiae

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