Vector systems for heterologous expression of proteins in Saccharomyces cerevisiae

Funk, Martin; Niedenthal, Rainer; Mumberg, Dominik; Brinkmann, Kay; Rönicke, Volker; Henkel, Thomas

doi:10.1016/s0076-6879(02)50967-8

Cited by 49 publications

(32 citation statements)

References 21 publications

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“…To investigate iron binding to Isa2 and Bio2 in vivo, the wild-type genes and the respective mutant alleles of ISA2 and BIO2 were inserted into the 2m plasmid p426-GPD downstream of the strong constitutive TDH3 promoter or into the low-copy plasmid p416-MET25 where expression is driven from the promoter of MET25 (13). The BIO2 alleles encoding proteins with defects in Fe/S cluster coordination were generated by site-directed mutagenesis using PCR.…”

Section: Methodsmentioning

confidence: 99%

The Iron-Sulfur Cluster Proteins Isa1 and Isa2 Are Required for the Function but Not for the De Novo Synthesis of the Fe/S Clusters of Biotin Synthase in Saccharomyces cerevisiae

et al. 2007

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The yeast Saccharomyces cerevisiae is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both isa mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of isa mutants to utilize desthiobiotin, since this defect was not cured by overexpression of BIO2. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.

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Section: Methodsmentioning

confidence: 99%

The Iron-Sulfur Cluster Proteins Isa1 and Isa2 Are Required for the Function but Not for the De Novo Synthesis of the Fe/S Clusters of Biotin Synthase in Saccharomyces cerevisiae

et al. 2007

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“…Plasmids p185 (pRS426.Rsc8.33HA) and p186 (pRS315.Rsc8.33HA), gifts from Marian Carlson, were described previously (Treich and Carlson 1997). Plasmids p935 (MET25pr.rsc7 248-435.133 Myc.TRP1) and p936 (MET25pr.rsc7 1-435.133Myc.TRP1) were constructed by cloning RSC7 fragments generated by PCR (using genomic DNA from YBC834) into the FB1521 plasmid (Funk et al 2002) using XhoI and XbaI or SpeI and BamHI, respectively.…”

Section: Methodsmentioning

confidence: 99%

The RSC Chromatin Remodeling Complex Bears an Essential Fungal-Specific Protein Module With Broad Functional Roles

et al. 2006

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RSC is an essential and abundant ATP-dependent chromatin remodeling complex from Saccharomyces cerevisiae. Here we show that the RSC components Rsc7/Npl6 and Rsc14/Ldb7 interact physically and/or functionally with Rsc3, Rsc30, and Htl1 to form a module important for a broad range of RSC functions. A strain lacking Rsc7 fails to properly assemble RSC, which confers sensitivity to temperature and to agents that cause DNA damage, microtubule depolymerization, or cell wall stress (likely via transcriptional misregulation). Cells lacking Rsc14 display sensitivity to cell wall stress and are deficient in the assembly of Rsc3 and Rsc30. Interestingly, certain rsc7D and rsc14D phenotypes are suppressed by an increased dosage of Rsc3, an essential RSC member with roles in cell wall integrity and spindle checkpoint pathways. Thus, Rsc7 and Rsc14 have different roles in the module as well as sharing physical and functional connections to Rsc3. Using a genetic array of nonessential null mutations (SGA) we identified mutations that are sick/ lethal in combination with the rsc7D mutation, which revealed connections to a surprisingly large number of chromatin remodeling complexes and cellular processes. Taken together, we define a protein module on the RSC complex with links to a broad spectrum of cellular functions.

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“…CAUTION PMSF is toxic; wear suitable protective clothing, gloves and eye/face protection. 0.1 M NADP + in ddH 2 O 20 mM cis-aconitic acid in ddH 2 O Triethanolamine buffer: 100 mM triethanolamine adjusted to pH 8 with 1 M NaOH IDH 40 U ml À1 in triethanolamine buffer with 10% (wt/vol) glycerol, store in small aliquots at À80 1C Isopropylmalate isomerase (Leu1) buffer: 20 mM Tris-Cl pH 7.4, 50 mM NaCl 10 mM DL-threo-3-isopropylmalic acid in ddH 2 O, dilute freshly from a 100 mM stock solution in ddH 2 O stored frozen at À20 1C Phosphate buffer: mix 1 M K 2 HPO 4 and 1 M KH 2 PO 4 in the right proportion to obtain pH 7.5 100 mM glucose-6-phosphate dipotassium salt in 20 mM phosphate buffer (pH 7.5) 100 U ml À1 glucose-6-phosphate dehydrogenase, dissolve 500 U in 5 ml of 0.1 M phosphate buffer (pH 7.5), store in small aliquots at À80 1C overproduction by substitution of the chromosomal promoter or expression from high copy plasmids utilizing suitable strong promoters (TDH3, GAL1-10 or MET25) 56 . The high natural concentration of iron in growth media and inside cells requires the depletion of ambient iron by cell cultivation at submicromolar iron concentrations ('iron poor' conditions) before addition of the radioisotope 10 .…”

Section: Experimental Designmentioning

confidence: 99%

Analysis of iron–sulfur protein maturation in eukaryotes

2009

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Vector systems for heterologous expression of proteins in Saccharomyces cerevisiae

Cited by 49 publications

References 21 publications

The Iron-Sulfur Cluster Proteins Isa1 and Isa2 Are Required for the Function but Not for the De Novo Synthesis of the Fe/S Clusters of Biotin Synthase in Saccharomyces cerevisiae

The Iron-Sulfur Cluster Proteins Isa1 and Isa2 Are Required for the Function but Not for the De Novo Synthesis of the Fe/S Clusters of Biotin Synthase in Saccharomyces cerevisiae

The RSC Chromatin Remodeling Complex Bears an Essential Fungal-Specific Protein Module With Broad Functional Roles

Analysis of iron–sulfur protein maturation in eukaryotes

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