Yeast Lysate Research Articles

Use of a two-hybrid system to identify mutations in Max that confer increased affinity for Myc.

A yeast two-hybrid system was used to identify mutants of Max that exhibit an increased affinity for Myc. Truncated forms of the Max helix-loop-helix/leucine zipper motif (HLH/Zip) were first expressed in a two- hybrid system in which the bait protein was the HLH/Zip motif of Myc. Deletion of amino acids both amino-terminal and carboxy-terminal to the leucine zipper of Max reduced Myc/Max heterodimer formation as evidenced by a 160-fold reduction in the expression of the lacZ gene. A library of partially randomized sequences encoding this minimal leucine zipper of Max was then screened using the two-hybrid system. Mutant forms of the Max leucine zipper were identified whose affinities for Myc, as measured by beta-galactosidase activity in yeast lysates, were from 8- to 200-fold greater than the wild-type Max zipper. These Max mutants were shown to interact specifically with Myc and not with wild-type Max. Of 29 mutants analyzed, all had a unique amino acid sequence. This result illustrates the value of a genetic screen in the identification of a collection of mutant forms of the Max leucine zipper whose structures would not have been predicted based on principles of structure-based design.

Recombinant Caspase-3 Expressed inPichia pastorisIs Fully Activated and Kinetically Indistinguishable from the Native Enzyme

Intracellular cysteine proteinases (caspases) play key roles in inflammation and apoptosis. Recombinant caspases are typically produced inEscherichia coliexpression systems with the attendant problems of solubilization, re-folding and activation of the protease. Here we describe the expression of hexahistidine-tagged caspase-3 (CPP32/Yama/Apopain) in the methylotropic yeastPichia pastoris,and the purification of soluble enzyme from yeast lysates using cobalt affinity chromatography. The recombinant protease is fully activated, stable, and cleaves the synthetic substrate DEVD-AFC (Km 16.8 μM) but not YVAD-AFC. It mediates the cleavage of the apoptotic death substrate poly(ADP-ribose) polymerase in cell extracts, but does not cleave pro-interleukin-1β. It is inhibited by the peptide DEVD-CHO (Ki2.2 nM), far less efficiently by YVAD-CMK (Ki0.3 μM), and not detectably by CrmA. By these criteria, recombinant caspase-3 is indistinguishable from native caspase-3 purified from apoptotic cell extracts. Activation of recombinant caspase-3 occurs in yeast in the absence of any intrinsic caspase activity, suggesting that caspase-3 can auto-activate. However, the purified enzyme was incapable of cleaving pro-caspase-3 indicating that autoactivation of caspase-3in vivois not likely to occur unless very high concentrations are achieved.

Biochemical characterization of the S135L allele of galactose‐1‐phosphate uridylyltransferase associated with galactosaemia

Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosaemia. The S135L mutation, which accounts for almost 50% of the GALT alleles in galactosaemia patients of African-American descent, has been associated with activities ranging from null to wild-type by different investigators examining cell lysates representing different tissues or model systems. Because of the crude nature of the lysates examined, however, and the absence of quantitative measures concerning GALT abundance in most of those lysates, the available data do not distinguish between differences in GALT enzyme expression/abundance, specific activity, or kinetic constants in these different tissues or systems. In an effort to overcome this uncertainty and investigate the biochemical impact of the S135L substitution on human GALT function under defined conditions, we have overexpressed both wild-type and S135L-mutant GALT sequences in a null-background yeast expression system, and purified both proteins to near homogeneity. Abundance of the wild-type and mutant proteins in crude yeast lysates differed by approximately 2-fold. Kinetic studies of the purified proteins, however, demonstrated that although K(m) values differed by < 2-fold, specific activities differed by 10-fold. Temperature-activity profiles revealed no significant differences, and coprecipitation studies demonstrated that S135L-hGALT subunits remained competent to self-associate in vivo. We conclude that the S135L substitution causes either steric or electrochemical changes sufficiently close to the active site in human GALT to result in partial impairment of the transferase reaction.

Immunological and chemical characterization of glycoproteins in IEF fractions of Blastomyces dermatitidis yeast lysate antigen.

After isoelectric focusing (IEF), fractions of a Blastomyces dermatitidis yeast lysate antigen were analysed for the presence of glycoproteins that may lead to cross-reactivity in immunoassays for the diagnosis of blastomycosis. Five major glycoproteins were apparent, two of which showed cross-reactivity when used in Western blots with sera obtained from dogs with histoplasmosis and coccidioidomycosis. These five glycoproteins were characterized for linkage to the proteins using N-glycosidase F (NGF) and for their lectin binding properties. The cross-reactive 235- and 160-kDa glycoproteins were found to possess mainly O-linked, high-mannose-type carbohydrates, and periodate-mediated oxidation of these molecules eliminated cross-reactivity observed with heterologous sera. Thus, the periodate-treated IEF antigens described here may be useful in solid-phase enzyme immunoassays for the diagnosis of blastomycosis.

A Rapid Method for the Preparation of Yeast Lysates That Facilitates the Immunodetection of Proteins Generated by the Yeast Two-Hybrid System

A Rapid Method for the Preparation of Yeast Lysates That Facilitates the Immunodetection of Proteins Generated by the Yeast Two-Hybrid System

PMR1, a Ca2+-ATPase in Yeast Golgi, Has Properties Distinct from Sarco/endoplasmic Reticulum and Plasma Membrane Calcium Pumps

PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to the Golgi, and shown to be required for normal secretory processes (Antebi, A., and Fink, G.R. (1992) Mol. Biol. Cell 3, 633-654). We provide biochemical evidence that PMR1 is a Ca2+-transporting ATPase in the Golgi, a hitherto unusual location for a Ca2+ pump. As a starting point for structure-function analysis using a mutagenic approach, we used the strong and inducible heat shock promoter to direct high level expression of PMR1 from a multicopy plasmid. Yeast lysates were separated on sucrose density gradients, and fractions assayed for organellar markers. PMR1 is found in fractions containing the Golgi marker guanosine diphosphatase, and is associated with an ATP-dependent, protonophore-insensitive 45Ca2+ uptake activity. This activity is virtually abolished in the absence of the expression plasmid. Furthermore, replacement of the active site aspartate within the phosphorylation domain had the expected effect of abolishing Ca2+ transport activity entirely. Interestingly, the mutant enzymes (Asp-371 --> Glu and Asp-371 --> Asn) demonstrated proper targeting to the Golgi, unlike analogous mutations in the related yeast H+-ATPase. Detailed characterization of calcium transport by PMR1 showed that sensitivity to inhibitors (vanadate, thapsigargin, and cyclopiazonic acid) and affinity for substrates (MgATP and Ca2+) were different from the previously characterized sarco/endoplasmic reticulum and plasma membrane Ca2+-ATPases. PMR1 therefore represents a new and distinct P-type Ca2+-ATPase. Because close homologs of PMR1 have been cloned from rat and other organisms, we suggest that Ca2+-ATPases in the Golgi will form a discrete subgroup that are important for functioning of the secretory pathway.

Translation-Competent Extracts fromSaccharomyces cerevisiae:Effects of L-A RNA, 5′ Cap, and 3′ Poly(A) Tail on Translational Efficiency of mRNAs

Yeast genetics has proven fruitful in the identification of key players that are involved in translational initiation. However, the exact roles of many translation initiation factors in translation initiation remain unknown. This has been due to lack of a suitablein vitrotranslation system in which the mode of action of certain translation factors can be studied. This report describes the preparation of cell-freeSaccharomyces cerevisiaelysates that can mediate the translation of exogenously added mRNAs. Optimal translation required the absence of viral L-A RNA in the lysate and the presence of both a 5′ cap and a 3′ poly(A) tail on the mRNAs. A cooperative effect of cap and poly(A) tail on translation initiation was observed, a property that has been found to operate in intact yeast cells as well. In addition, the yeast lysates mediated translational initiation through several viral internal ribosome entry sites, demonstrating that the yeast translation apparatus can perform internal initiation. Thus, these lysates may be useful in the biochemical analysis of cap-dependent and cap-independent translation events.

In vivo analysis of the Hsp90 cochaperone Sti1 (p60).

Hsp90 interacts with Sti1 (p60) in lysates of yeast and vertebrate cells. Here we provide the first analysis of their interaction in vivo. Saccharomyces cerevisiae mutations that eliminate Sti1 or reduce intracellular concentrations of Hsp90 individually have little or no effect on growth at normal temperatures. However, when combined, the mutations greatly reduce or eliminate growth. Furthermore, overexpression of Sti1 has allele-specific effects on cells carrying various hsp90ts point mutations. These genetic interactions provide strong evidence that Hsp90 and Sti1 interact in vivo and that their functions are closely allied. Indeed, deletion of STI1 reduces the in vivo activity of the Hsp90 target protein, glucocorticoid receptor (GR). Mutations in GR that eliminate interaction with Hsp90 also eliminate the effects of the sti1 deletion. Examination of GR protein complexes in the sti1 deletion mutant reveals a selective increase in the concentration of GR-Ydj1 complexes, supporting previous hypotheses that Ydj1 functions at an early step in the maturation of GR and that Sti1 acts at an intermediate step. Deletion of STI1 also reduces the in vivo activity of another, unrelated Hsp90 target protein, v-Src. Our data indicate that Sti1 is a general factor in the maturation of Hsp90 target proteins and support earlier suggestions that Hsp90 matures even very different target proteins by a similar mechanism.

The refolding activity of the yeast heat shock proteins Ssa1 and Ssa2 defines their role in protein translocation.

Ssa1/2p, members of one of the yeast cytosolic hsp70 subfamilies, have been implicated in the translocation of secretory proteins into the lumen of the ER. The involvement of these hsp70s in translocation was tested directly by examining the effect of immunodepleting Ssa1/2p from yeast cytosol and subsequently testing the cytosol for its ability to support co- and post-translational translocation of prepro-alpha-factor. Depletion of Ssa1/2p had no effect on the efficiency of translocation in this in vitro assay. The system was used to examine the effect of the absence of Ssa1/2p on two other putative hsp70 functions: cotranslational folding of nascent luciferase and refolding of denatured luciferase. Depletion of Ssa1/2p had no effect on the ability of the yeast lysate to synthesize enzymatically active luciferase, but had a dramatic effect on the ability of the lysate to refold chemically denatured luciferase. These results demonstrate, for the first time, the refolding activity of Ssa1/2p in the context of the yeast cytosol, and define refolding activity as a chaperone function specific to Ssa1/2p, aprt from other cytosolic hsp70s. They also suggest that Ssa1/2p do not play a significant role in chaperoning the folding of nascent polypeptides. The implications of these findings for Ssa1/2p activity on their proposed role in the process of translocation are discussed.

Synthesis of a chemiluminescent probe useful for the purification of steroid 5α-reductase

Steroid 5α-reductase is a system of two isozymes (5αR-1 and 5αR-2), which is related to several human diseases. The synthesis of a chemiluminescent probe (AZA-LU) useful for purification of 5αR is described. AZA-LU has a chemiluminescence detectable up to 10 −15 M and is a good inhibitor (IC 50 = 0.5 μM) for rat 5αR-1 expressed in transformed Saccharomicyes cerevisiae yeast. Incubation of AZA-LU with yeast lysates, followed by SDS-PAGE, led to the detection of two luminescent protein regions with estimated molecular weights of 26 kDa and 21 kDa.

Regulation of Chromosome Segregation by Glc8P, a Structural Homolog of Mammalian Inhibitor 2 That Functions as both an Activator and an Inhibitor of Yeast Protein Phosphatase 1

The Ipl1 protein kinase is essential for proper chromosome segregation and cell viability in the budding yeast Saccharomyces cerevisiae. We have previously shown that the temperature-sensitive growth phenotype of conditional ipl1-1ts mutants can be suppressed by a partial loss-of-function mutation in the GLC7 gene, which encodes the catalytic subunit (PP1C) of protein phosphatase 1, thus suggesting that this enzyme acts in opposition to the Ipl1 protein kinase in regulating yeast chromosome segregation. We report here that the Glc8 protein, which is related in primary sequence to mammalian inhibitor 2, also participates in this regulation. Like inhibitor 2, the Glc8 protein is heat stable, exhibits anomalous electrophoretic mobility, and functions in vitro as an inhibitor of yeast as well as rabbit skeletal muscle PP1C. Interestingly, overexpression as well as deletion of the GLC8 gene results in a partial suppression of the temperature-sensitive growth phenotype of ipl1ts mutants and also moderately reduces the amount of protein phosphatase 1 activity which is assayable in crude yeast lysates. In addition, the chromosome missegregation phenotype caused by an increase in the dosage of GLC7 is totally suppressed by the glc8-delta 101::LEU2 deletion mutation. These findings together suggest that the Glc8 protein is involved in vivo in the activation of PP1C and that when the Glc8 protein is overproduced, it may also inhibit PP1C function. Furthermore, site-directed mutagenesis studies of GLC8 suggest that Thr-118 of the Glc8 protein, which is equivalent to Thr-72 of inhibitor 2, may play a central role in the ability of this protein to activate and/or inhibit PP1C in vivo.

The Yeast Proprotein Convertase Encoded by YAP3 Is a Glycophosphatidylinositol-anchored Protein That Localizes to the Plasma Membrane

The yeast YAP3 gene encodes an aspartyl endoprotease that cleaves precursor proteins at selected pairs of basic amino acids and after single arginine residues. Biosynthetic studies of this proprotein processing enzyme indicate that Yap3 is predominantly cell-associated and migrates as a approximately 160-kDa protein on SDS-polyacrylamide gel electrophoresis. Nearly equal amounts of Yap3 are immunodetected in a-haploid, alpha-haploid, and a/alpha-diploid yeast, demonstrating that the expression of YAP3 is not mating type-specific. As shown by endoglycosidase H treatment, which drastically reduces both the estimated molecular mass and the heterogeneity of the protein on SDS-polyacrylamide gel electrophoresis (68 versus 160 kDa), the oligosaccharides N-linked to the protein are subjected to extensive outer chain mannosylation. Outer chain sugar mannosylation takes place in the Golgi apparatus and is commonly found on yeast secreted glycoproteins and/or cell wall mannoproteins. Treatment of the total yeast membranes with chemical agents known to disrupt protein-protein and protein-lipid interactions reveal that Yap3 is membrane-associated. Based upon the release of the membrane-bound form by bacterial phosphatidylinositol phospholipase C digestion and metabolic labeling of the protein with myo-[3H]inositol, Yap3 owes its association with the membrane to the addition of a glycophosphatidylinositol anchor. The cellular localization of Yap3 has been addressed by subcellular fractionation studies. In both differential centrifugation of intracellular organelles and sucrose density gradients, the bulk of Yap3 at steady state co-localizes with the plasma membrane azide-insensitive ATPase. Furthermore, consistent with the transport of Yap3 to the plasma membrane, the endoprotease sediments with secretory vesicles which accumulate at restrictive temperature in the late secretory mutant sec1-1. We therefore conclude that the endoprotease encoded by YAP3 is a glycophosphatidylinositol-anchored protein, which can process substrates both intracellularly and at the cell surface.

Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the products of at least 14 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Two of these genes, SEC8 and SEC15, encode components of a 1-2-million D multi-subunit complex that is found in the cytoplasm and associated with the plasma membrane. In this study, oligonucleotide-directed mutagenesis is used to alter the COOH-terminal portion of Sec8 with a 6-histidine tag, a 9E10 c-myc epitope, or both, to allow the isolation of the Sec8/15 complex from yeast lysates either by immobilized metal affinity chromatography or by immunoprecipitation. Sec6 cofractionates with Sec8/15 by immobilized metal affinity chromatography, gel filtration chromatography, and by sucrose velocity centrifugation. Sec6 and Sec15 coimmunoprecipitate from lysates with c-myc-tagged Sec8. These data indicate that the Sec8/15 complex contains Sec6 as a stable component. Additional proteins associated with Sec6/8/15 were identified by immunoprecipitations from radiolabeled lysates. The entire Sec6/8/15 complex contains at least eight polypeptides which range in molecular mass from 70 to 144 kD. Yeast strains containing temperature sensitive mutations in the SEC genes were also transformed with the SEC8-c-myc-6-histidine construct and analyzed by immunoprecipitation. The composition of the Sec6/8/15 complex is disrupted specifically in the sec3-2, sec5-24, and sec10-2 strain backgrounds. The c-myc-Sec8 protein is localized by immunofluorescence to small bud tips indicating that the Sec6/8/15 complex may function at sites of exocytosis.

Mutations Causing Coagulation Factor XIII Subunit A Deficiency: Characterization of the Mutant Proteins After Expression in Yeast

We identified the mutations causing factor XIII A subunit deficiency in two families. Two distinct mutations were identified in the S family: the nonsense mutation Tyr 441-->stop in exon 11, inherited through the paternal line, and the missense mutation Asn 60-->Lys in exon 3, inherited through the maternal line. Two members of the J family were heterozygous for the previously described type 3 A subunit. The substitution giving rise to the type 3 variant was found to be Gly 501-->Arg in exon 12. The Asn 60-->Lys and Gly 501-->Arg mutations were constructed in cDNA clones and expressed in yeast (Saccharomyces cerevisiae AH22). Although mRNA could be detected, protein containing the Asn 60-->Lys substitution could not be detected, suggesting extreme instability or susceptibility to proteolysis. A subunits containing the Gly 501-->Arg substitution were expressed and found to be enzymatically active in fresh yeast lysates. This variant has thermal instability and lost activity during storage or purification. Gel filtration studies suggested that the type 3 variant assembled as a dimer, as do normal A subunits. The data suggest that the Gly 501-->Arg (Type 3 variant) would cause severe factor XIII deficiency if inherited in the homozygous form or as a compound heterozygote with another deleterious mutation.

Effect on humoral tolerance (IgG and IgE) in dogs by oral administration of ovalbumin and Der p I

The effect of oral administration of ovalbumin (OVA) or recombinant house dust mite allergen ( Der p I) to dogs upon specific IgG and IgE reactions to subcutaneous immunization with these antigens was studied. Daily feeding of 10 × 10 g of OVA resulted in a non-responsiveness to subsequent parenteral immunization with OVA in two young dogs. The same two dogs were also immunized parenterally with Der p I and showed a pronounced IgG response against native Der p I, confirming that the non-responsiveness to OVA was antigen-specific. Thus, it has been demonstrated that it is possible to induce oral tolerance in dogs. Two other dogs of the same litter that received 2 × 10 mg of recombinant Der p I in a crude yeast lysate per os reacted to immunication with OVA with pronounced IgG and IgE production against OVA, further confirming the antigen-specificity of the OVA tolerance. However, tolerance to Der p I was not induced, as evidenced by a strong IgG response to immunization after per os application of the antigen, possibly because the oral dose was too small.

Localization of a protein a‐tagged kex2 protein to the vacuole of Saccharomyces cerevisiae allows rapid purification of vacuolar membranes

We have previously reported an immunoisolation procedure which allows purification of Kex2p-containing Golgi membranes from lysed yeast cells. In order to evaluate the use of tagging procedures in organelle isolation we set out to isolate the same Golgi membrane fraction using a version of the Kex2 protease that had been affinity-tagged at its C-terminus. This protein is found to be localized in the vacuole, providing the basis of a method for the affinity-purification of vacuolar membranes.

Induction and detection of delayed dermal hypersensitivity in guinea-pigs immunized withBlastomyces dermatitidislysate and filtrate antigens

Guinea-pigs were immunized using yeast phase antigens (lysate and filtrate preparations) from two strains of Blastomyces dermatitidis (T-58 and Le). Following a sensitization period, the animals were skin tested on days 40 and 216 using T-58 and Le yeast and mycelial phase lysate and filtrate antigen preparations for the detection of delayed dermal hypersensitivity (DTH). Using the Friedman's analysis of variance by rank test, significant differences were found in the efficacy of the immunogens to induce DTH in the animals when skin tested on both occasions (P < 0.05). Optimal reactivity was observed in guinea-pigs immunized with Le yeast lysate (mean axes of induration ranging from 12.0 to 18.8 mm and 7.0 to 18.5 mm on day 40 and 216, respectively) and T-58 yeast filtrate (mean axes of induration ranging from 7.5 to 18.0 mm and 8.0 to 17.0 mm on day 40 and 216, respectively) when the immunogens were administered with adjuvant. When the same data was analysed using the Friedman's test with regard to evaluating the efficacy of the skin test antigens to detect DTH, significant differences were found between them (P < 0.05) with optimal results using the Le mycelial filtrate and yeast lysate antigens (mean axes of induration ranging from 7.0 to 16.5 mm and 7.5 to 14.5 mm, respectively) when skin testing was done on day 40. When skin testing was done on day 216, the T-58 and Le mycelial lysate antigens gave optimal results (mean axes of induration ranging from 9.0 to 18.5 mm and 7.5 to 15.0 mm, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and Characterization of Two Monoclonal Antibodies Raised Against tmsl Protein of Fission Yeast

Monoclonal antibodies were produced against recombinant tms1 protein of fission yeast. The antibodies of IgG3 subclass were isolated from serum-free cell culture medium and purified by affinity chromatography on protein A-Sepharose. The antibodies can be used to detect specifically the tms1 protein on immunoblots of total yeast lysates. In addition, native tms1 protein is specifically precipitated by these antibodies from yeast lysates.

Expression, Purification, and Characterization of Porcine Leukocyte 12-Lipoxygenase Produced in the Methylotrophic Yeast, Pichia pastoris

A cDNA coding for porcine leukocyte 12-lipoxygenase was expressed intracellularly in the methylotrophic yeast Pichia pastoris under the regulatory control of the alcohol oxidase promoter. The recombinant 12-lipoxygenase contained in the yeast cell lysate was soluble, displayed the catalytic properties of the native enzyme, and was recognized by antibodies prepared against native 12-lipoxygenase derived from porcine leukocytes. The catalytically active enzyme of the 100,000 x g supernatant obtained from the yeast lysate was readily purified by immunoaffinity chromatography to near homogeneity. Porcine leukocyte 12-lipoxygenase is the first arachidonic acid oxygenase to be expressed in yeast, an easy, inexpensive, and rapid method of expressing native and site-directed mutants of recombinant proteins.

Vectors for Cu(2+)-inducible production of glutathione S-transferase-fusion proteins for single-step purification from yeast.

We describe six new yeast episomal vectors which encode glutathione S-transferase (GST) affinity tags. These allow for the production of GST-fusion proteins in Saccharomyces cerevisiae under the control of the CUP1 promoter. Affinity chromatography with glutathione-Sepharose permits convenient purification of the fusion protein from a yeast lysate. The presence of a protease cleavage site facilitates subsequent removal of the GST tag. The expression and single-step purification of both GST and a functional GST-metallothionein fusion from yeast are shown as an example of the application of these vectors.