Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression

Mumberg, Dominik; Müller, Rolf; Funk, Martin

doi:10.1093/nar/22.25.5767

Cited by 903 publications

(809 citation statements)

References 10 publications

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“…The cDNAs for full-length CYP2E1, ϩ5/1A1, ϩ331A1, and su9-DHFR were cloned into the 2-vector, pTEF-URA3, or the centromeric pTEF-URA3 plasmid (37).…”

Section: Methodsmentioning

confidence: 99%

An Unusual TOM20/TOM22 Bypass Mechanism for the Mitochondrial Targeting of Cytochrome P450 Proteins Containing N-terminal Chimeric Signals

Anandatheerthavarada

Sepuri²,

Biswas³

et al. 2008

Journal of Biological Chemistry

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Previously we showed that xenobiotic-inducible cytochrome P450 (CYP) proteins are bimodally targeted to the endoplasmic reticulum and mitochondria. In the present study, we investigated the mechanism of delivery of chimeric signal-containing CYP proteins to the peripheral and channel-forming mitochondrial outer membrane translocases (TOMs). CYP؉33/1A1 and CYP2B1 did not require peripheral TOM70, TOM20, or TOM22 for translocation through the channel-forming TOM40 protein. In contrast, CYP؉5/1A1 and CYP2E1 were able to bypass TOM20 and TOM22 but required TOM70. CYP27, which contains a canonical cleavable mitochondrial signal, required all of the peripheral TOMs for its mitochondrial translocation. We investigated the underlying mechanisms of bypass of peripheral TOMs by CYPs with chimeric signals. The results suggested that interaction of CYPs with Hsp70, a cytosolic chaperone involved in the mitochondrial import, alone was sufficient for the recognition of chimeric signals by peripheral TOMs. However, sequential interaction of chimeric signal-containing CYPs with Hsp70 and Hsp90 resulted in the bypass of peripheral TOMs, whereas CYP27 interacted only with Hsp70 and was not able to bypass peripheral TOMs. Our results also show that delivery of chimeric signal-containing client proteins by Hsp90 required the cytosol-exposed N-terminal 143 amino acids of TOM40. TOM40 devoid of this domain was unable to bind CYP proteins. These results suggest that, compared with the unimodal mitochondria-targeting signals, the chimeric mitochondriatargeting signals are highly evolved and dynamic in nature.Protein targeting to different subcellular compartments is directed by specific N-terminal or internal signals, which serve as destination-specific mail delivery codes (1, 2). The signal sequences of proteins targeted to different membrane compartments, such as the endoplasmic reticulum (ER), 2 peroxisomes, and mitochondria, vary markedly in terms of amino acid sequence, hydrophobicity, and secondary structure and interact with distinctly different sets of carrier proteins, receptors, and protein translocator complexes (3-8). These observations have led to a widely accepted view that most protein targeting signals are unimodal in nature, which in turn restricts the destination of a given protein to a single subcellular compartment.ER-targeted proteins contain a distinct N-terminal hydrophobic signal for binding to a signal recognition particle, which in turn targets the emerging nascent chains to the ER (9). With certain exceptions (1, 10), ER targeting is thought to be cotranslational. Current models of protein targeting imply that the ER or mitochondrial destination of a protein is determined at the pretranslational level by virtue of the signal sequence that the protein carries. Many mitochondria-and ER-targeted proteins are encoded by a distinct set of genes. In a limited number of cases, characteristic mitochondrial targeting sequences are generated by differential expression of the gene using either alternate transcription/tr...

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“…The cDNAs for full-length CYP2E1, ϩ5/1A1, ϩ331A1, and su9-DHFR were cloned into the 2-vector, pTEF-URA3, or the centromeric pTEF-URA3 plasmid (37).…”

Section: Methodsmentioning

confidence: 99%

An Unusual TOM20/TOM22 Bypass Mechanism for the Mitochondrial Targeting of Cytochrome P450 Proteins Containing N-terminal Chimeric Signals

Anandatheerthavarada

Sepuri²,

Biswas³

et al. 2008

Journal of Biological Chemistry

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“…Allele-specific N-terminal 3xFLAG-QNS1 fusions were created by PCR amplification of the QNS1 alleles from plasmids pB177, pB178, and pB251 with primers 7119 and 7120. Products of these reactions were used as templates for PCR with primers 7118 and 7120, restricted with BamHI and EcoRI, and cloned into vectors p413GAL1 and p414GAL1 (21), respectively, to generate plasmids pB320, pB326, and pB324. Allele-specific 2 ϫ HA-QNS1 fusions were similarly made from plasmids pB177 and pB251 as templates, primers 7117 and 7120 in the first PCR and primers 7116 and 7120 in the second PCR.…”

Section: Methodsmentioning

confidence: 99%

“…Allele-specific 2 ϫ HA-QNS1 fusions were similarly made from plasmids pB177 and pB251 as templates, primers 7117 and 7120 in the first PCR and primers 7116 and 7120 in the second PCR. Restricted products were cloned into vectors p413GAL1 and p414GAL1 (21), respectively, to generate pB321 and pB325. Plasmids pB341, pB342, and pB343 were created by side-directed mutagenesis of plasmid pB326 with a mixture of primers 7039 and 7040 to create E45A and/or K114A mutations in addition to the C175A mutation of plasmid pB326.…”

Section: Methodsmentioning

confidence: 99%

Eukaryotic NAD+ Synthetase Qns1 Contains an Essential, Obligate Intramolecular Thiol Glutamine Amidotransferase Domain Related to Nitrilase

Bieganowski¹,

Pace²,

Brenner³

2003

Journal of Biological Chemistry

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NAD ؉ is an essential co-enzyme for redox reactions and is consumed in lysine deacetylation and poly(ADPribosyl)ation. NAD ؉ synthetase catalyzes the final step in NAD ؉ synthesis in the well characterized de novo, salvage, and import pathways. It has been long known that eukaryotic NAD ؉ synthetases use glutamine to amidate nicotinic acid adenine dinucleotide while many purified prokaryotic NAD ؉ synthetases are ammoniadependent. Earlier, we discovered that glutamine-dependent NAD ؉ synthetases contain N-terminal domains that are members of the nitrilase superfamily and hypothesized that these domains function as glutamine amidotransferases for the associated synthetases. Here we show yeast glutamine-dependent NAD ؉ synthetase Qns1 requires both the nitrilase-related active-site residues and the NAD ؉ synthetase active-site residues for function in vivo. Despite failure to complement the lethal phenotype of qns1 disruption, the former mutants retain ammonia-dependent NAD ؉ synthetase activity in vitro, whereas the latter mutants retain basal glutaminase activity. Moreover, the two classes of mutants fail to trans-complement despite forming a stable heteromultimer in vivo. These data indicate that the nitrilaserelated domain in Qns1 is the fourth independently evolved glutamine amidotransferase domain to have been identified in nature and that glutamine-dependence is an obligate phenomenon involving intramolecular transfer of ammonia over a predicted distance of 46 Å from one active site to another within Qns1 monomers.Nicotinamide-adenine dinucleotide (NAD ϩ ) 1 and its phosphorylated form NADP are essential for oxidizing reactions in the cell, whereas reduced forms of these co-enzymes, NADH and NADPH, are essential for supplying reducing equivalents. Beyond the reversible functions of NAD ϩ in hundreds of enzymedependent redox reactions, at least two types of enzymes consume NAD ϩ in eukaryotic nuclei in the process of forming or reversing post-translational modifications. Poly(ADP-ribose) polymerases respond to DNA strand breaks by transferring the ADP-ribose moiety of NAD ϩ to target proteins, thereby facilitating DNA repair and other processes (1, 2). Sirtuins, a family of protein lysine-deacetylases related to yeast Sir2, reverse regulatory acetyl modification of lysines on histones, p53, and other proteins typically to alter the assembly of nucleoprotein complexes (3, 4). NAD ϩ is consumed by Sir2 to produce a mixture of 2Ј-and 3Ј-O-acetylated ADP-ribose plus nicotinamide and the deacetylated polypeptide (5). NAD ϩ -dependent deacetylation reactions are required not only for alterations in gene expression but also for repression of ribosomal DNA recombination and extension of lifespan in response to calorie restriction (6, 7). In prokaryotes, Sirtuins perform lysine deacetylation to alter chromatin structure (8) as well as to regulate acetyl-CoA synthetase and potentially other metabolic enzymes (9).Interest in the biological functions of Sir2 and Sirtuins has lead to re-examination of the biosynthetic ro...

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“…The cDNA sequence encoding the Z variant of ␣-1 protease inhibitor (A1PiZ) (McCracken and Kruse, 1993) was cloned either into vectors containing the inducible GAL1 promoter: pYES2.0 (2 , Amp r , URA3; Invitrogen) and pBM743 (CEN/ARS, Amp r , URA3; Invitrogen), or into a vector with the repressible MET25 promoter (Mumberg et al, 1994): p426MET25 (2 , Amp r , URA3; ATCC, Manassas, VA). Triple-hemagglutinin (HA)-tagged cystic fibrosis transmembrane conductance regulator (CFTR) expression in yeast was driven by the constitutive phosphoglycerate kinase promoter in a 2 plasmid (Zhang et al, 2002).…”

Section: Strains Plasmids Media and Antiseramentioning

confidence: 99%

Characterization of anERADGene asVPS30/ATG6Reveals Two Alternative and Functionally Distinct Protein Quality Control Pathways: One for Soluble Z Variant of Human α-1 Proteinase Inhibitor (A1PiZ) and Another for Aggregates of A1PiZ

Kruse

Brodsky

McCracken

2006

MBoC

188

185

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The Z variant of human ␣-1 proteinase inhibitor (A1PiZ) is a substrate for endoplasmic reticulum-associated protein degradation (ERAD). To identify genes required for the degradation of this protein, A1PiZ degradation-deficient (add) yeast mutants were isolated. The defect in one of these mutants, add3, was complemented by VPS30/ATG6, a gene that encodes a component of two phosphatidylinositol 3-kinase (PtdIns 3-kinase) complexes: complex I is required for autophagy, whereas complex II is required for the carboxypeptidase Y (CPY)-to-vacuole pathway. We found that upon overexpression of A1PiZ, both PtdIns 3-kinase complexes were required for delivery of the excess A1PiZ to the vacuole. When the CPY-to-vacuole pathway was compromised, A1PiZ was secreted; however, disruption of autophagy led to an increase in aggregated A1PiZ rather than secretion. These results suggest that excess soluble A1PiZ transits the secretion pathway to the trans-Golgi network and is selectively targeted to the vacuole via the CPY-to-vacuole sorting pathway, but excess A1PiZ that forms aggregates in the endoplasmic reticulum is targeted to the vacuole via autophagy. These findings illustrate the complex nature of protein quality control in the secretion pathway and reveal multiple sites that recognize and sort both soluble and aggregated forms of aberrant or misfolded proteins. INTRODUCTIONCell function and survival depend on protein quality control to identify and remove aberrant proteins. Although two cellular sites of proteolysis are known, the lysosome/vacuole and cytoplasmic 26S proteasome, the recognition of aberrant proteins and mechanisms for delivery to these sites are still being defined. Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control process in which aberrant or misassembled proteins in the secretory pathway are identified and removed (reviewed in Hampton, 2002;Tsai et al., 2002;Kostova and Wolf, 2003;McCracken and Brodsky, 2003). After entering the endoplasmic reticulum (ER), a nascent protein that fails to fold or assemble properly can be "recognized" by the ER quality control machinery, retained within the ER, and then retrotranslocated to the cytoplasm where it is degraded by the proteasome.The recognition of ERAD substrates must exhibit flexibility to distinguish slowly folding proteins from aberrant proteins. Molecular chaperones play a critical role in this selection process; for example, the ER heat-shock protein (Hsp70), BiP, is vital for the selection of soluble substrates and is believed to hold the substrates in an unfolded state competent for retrotranslocation (Nishikawa et al., 2001;Kabani et al., 2003).The complexity of the ERAD pathway has emerged with the identification of new ERAD substrates and the components required for their selection and degradation in yeast. For example, the analysis of topologically distinct ERAD substrates indicates that molecular chaperones in the cytoplasm and in the ER lumen distinguish membrane and soluble substrates, respectively (Huyer et al., 20...

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Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression

Cited by 903 publications

References 10 publications

An Unusual TOM20/TOM22 Bypass Mechanism for the Mitochondrial Targeting of Cytochrome P450 Proteins Containing N-terminal Chimeric Signals

An Unusual TOM20/TOM22 Bypass Mechanism for the Mitochondrial Targeting of Cytochrome P450 Proteins Containing N-terminal Chimeric Signals

Eukaryotic NAD+ Synthetase Qns1 Contains an Essential, Obligate Intramolecular Thiol Glutamine Amidotransferase Domain Related to Nitrilase

Characterization of anERADGene asVPS30/ATG6Reveals Two Alternative and Functionally Distinct Protein Quality Control Pathways: One for Soluble Z Variant of Human α-1 Proteinase Inhibitor (A1PiZ) and Another for Aggregates of A1PiZ

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