Expression of a cDNA derived from the yeast killer preprotoxin gene: implications for processing and immunity.

Hanes, Steven D.; Burn, Virginia E.; Sturley, Stephen L.; Tipper, Donald J.; Bostian, Keith A.

doi:10.1073/pnas.83.6.1675

Cited by 45 publications

(29 citation statements)

References 21 publications

(50 reference statements)

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“…The role of the alpha polypeptide in S. cerevisiae kl toxin (4,19,45) is the interaction with the cell membrane that kills the cell by disrupting ion transport (8,12,23). This is similar to the mode of action of the cytotoxins (25,38 The primary sequence similarity is most evident in the region of residues 26 to 45 of KP6 beta (Fig.…”

Section: Discussionmentioning

confidence: 88%

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Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

Tao¹,

Ginsberg²,

Banerjee³

et al. 1990

Mol. Cell. Biol.

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There are a number of yeasts that secrete killer toxins, i.e., proteins lethal to sensitive cells of the same or related species. Ustilago maydis, a fungal pathogen of maize, also secretes killer toxins. The best characterized of the U. maydis killer toxins is the KP6 toxin, which consists of two small polypeptides that are not covalently linked. In this work, we show that both are encoded by one segment of the genome of a double-stranded RNA virus. They are synthesized as a preprotoxin that is processed in a manner very similar to that of the Saccharomyces cerevisiae kl killer toxin, also encoded by a double-stranded RNA virus. Active U. maydis KP6 toxin was secreted from S. cerevisiae transformants expressing the KP6 preprotoxin. The two secreted polypeptides were not glycosylated in U. maydis, but one was glycosylated in S. cerevisiae. Comparison of known and predicted cleavage sites among the five killer toxins of known sequence established a threeamino-acid specificity for a KEX2-like enzyme and predicted a new, undescribed processing enzyme in the secretory pathway in the fungi. The mature KP6 toxin polypeptides had hydrophobicity profiles similar to those of other known cellular toxins.Killer toxins are known in eight genera of yeasts (53).Those of Saccharomyces cerevisiae, Picahia kluyveri, and Kluyv'eromyces lactis are best characterized (12,23,46). In S. cerevisiae, the kl toxin consists of two subunits, alpha (103 amino acids) and beta (83 amino acids), joined by two disulfide bonds (5,42,54). These are encoded on one double-stranded RNA (dsRNA) of the S. cerev'isiae virus (ScV) and synthesized as a prepropolypeptide of 317 amino acids, which is cleaved by signal peptidase, the KEXi and KEX2 proteases, and by at least one more unspecified protease during its maturation (5,9,15,17,22,42,54 P6, respectively (24, 26-30). Their mode of action is unknown, but like the S. cerevisiae kl toxin, the KP6 toxin has cell wall receptors (44). Unlike the ScV kl toxin, in which the two subunits are joined by disulfide bonds (5), or the K. lac tis toxin, in which the three subunits are present in a (probably) noncovalently bonded complex (43), the KP6 toxin polypeptides interact with the cell independently, as monomers (37). Genetic and in vitro translation data did not indicate conclusively whether the two KP6 polypeptides were synthesized as one preprotoxin or as two (28,37).We have shown that the KP6 toxin polypeptides are synthesized as one preprotoxin of 219 amino acids with a typical signal sequence. The preprotoxin is cleaved by an enzyme with specificity similar to that of the S. cerevisiae KEX2 enzyme (22) and by at least one other enzyme with specificity like that of the enzyme which cleaves off the N terminus of the S. cerevisiae kl preprotoxin. The result is two secreted polypeptides alpha (78 amino acids) and beta (81 Amino acids), whose C termini are predicted in analogy with the kl toxin. The KP6 preprotoxin was properly processed in S. cerev-isiae, and active KP6 alpha and beta were secreted. KP6 alp...

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Section: Discussionmentioning

confidence: 88%

“…The first ATG of the open reading frame is the first ATG of the plus strand. The predicted N terminus has a typical sequence for a signal polypeptide, with a predicted signal peptidase cleavage site after the alanine at position 19 (50). The portion of the predicted polypeptide just C terminal to the signal sequence is very rich in cysteine.…”

Section: Methodsmentioning

confidence: 99%

Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

Tao¹,

Ginsberg²,

Banerjee³

et al. 1990

Mol. Cell. Biol.

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“…The 82-1 construction resulted in the indicated change in the predicted sequence of the amino terminus, from NH2-Met-Arg-Phe (MRF) in native prepro-afactor to NH2-Met-Phe-Lys (MFK) in 82-1. thesis and membrane translocation prevents completion of translation of secretory proteins in the cytoplasmic compartment. In yeast, however, unglycosylated forms of several glycoproteins have been detected (16,17,18); in all cases, this phenomenonis attributed to overload of the secretory apparatus, due to elevated levels of expression of the genes from multicopy plasmid. An alternative explanation is that the p82-l encoded hybrid might be a poor substrate relative to native pro-a-factor for some component of the RERand that a sequence specific defect might identify topogenic domains in proa-factor which are altered in the hybrids.…”

Section: Resultsmentioning

confidence: 99%

Topogenic Effect of Positively Charged N-terminal Amino Acid in ER Translocation of Yeast .ALPHA.-Factor Precursor.

Lee¹,

Cheong²,

Choe

et al. 1996

Cell Struct. Funct.

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ABSTRACT. Expression of fusion proteins between prepro-a-factor and somatostatin (SRIF) in yeast, resulted in the correct processing and secretion of the heterologous 14-amino acid SRIF peptide (1). Whenthe chimeric genes were placed under the control of yeast acid phosphatase (PHO5) promoter, significant amount of an unglycosylated form of the fusion precursor molecule accumulated intracellularly, suggesting disruption of an endoplasmic reticulum-mediated function. Wereport here that the appearance of the precursor is due to an alteration in the three amino terminal residues of the chimera, i.e., Met-Arg-Phe in native prepro-a-factor is changed to Met-Phe-Lys in the hybrids. The unglycosylated precursor represents a population of molecules that are disrupted at an early stage of targeting to or translocation across the endoplasmic reticulum membrane. Our data demonstrate that the N-terminus plays an important role in topogenesis. Furthermore, these results show that translocation and glycosylation can be uncoupled from protein synthesis in vivo, and therefore can be posttranslational events in yeast.The molecular determinants of protein transit through the secretory pathway are becoming increasingly well understood (2). Elucidation of the signal recognition particle (SRP) cycle in mammalian cells has clarified how the RERmembraneis selected from other cellular membranesfor initiation of the secretory process (3, 4), but has not addressed how proteins are subsequently translocated across the lipid bilayer and sorted prior to secretion. The precursors to the small peptide hormones are proving to be excellent models to investigate these processes (5, 6). Yeast prepro-a-factor, for example, contains a signal sequence of 19 amino acids that is cleaved in vivo and in vitro (7, 8). The signal is followed by a pro region of 63 amino acids, containing three consensus sites for N-linked glycosylation; at the carboxyl terminus are four repeats of the 13 residue afactor peptide, separated by spacers that include endoproteolytic processing sites. Prepro-a-factor is unique among native presecretory proteins in that it can be efficiently translocated across yeast microsomal membranes in a post-translational manner (8, 9, 10). This in vitro reaction is specific for yeast membranes and requires energy and one or more of a family of cytosolic yeast heat shock proteins (hsp70s). Though it is unclear to what extent such post-translational uptake occurs in vivo, these results raise the possibility that prepro-afactor may attain significant tertiary structure prior to membrane interaction. To identify topogenic domains in the prepro-a-factor, we have constructed a series of fusion between prepro-a-factor and preprosomatostatin, the precursor to the vertebrate tetradecapeptide hormonesomatostatin (SRIF). PreproSRIF contains a typical signal sequence, which is cleaved cotranslationally (1 1), and a pro region of 82 amino acids preceding the mature hormone sequence (12). A series of recombinant DNAmolecules was constructed that encodes...

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“…Strains containing one (EGY18), two (EGY23), or three (EGY38) colEI operators upstream of the single chromosomal LEU2 gene were isolated. These strains were made his3 Ϫ by being mated to the strain GG100-14D (MAT␣ his3 trp1 pho5) (32), with selection for LEU ϩ HIS ϩ diploids, sporulation, and selection for random spore products that were leu2, ura3, trp1, his3, and GAL ϩ . EGY48, EGY195, and EGY191 are derivatives of EGY38, EGY22, and EGY18, respectively.…”

Section: Cloning and Bacterial Strains Escherichia Colimentioning

confidence: 99%

Correlation of Two-Hybrid Affinity Data with In Vitro Measurements

Estojak

Brent

Golemis

1995

Molecular and Cellular Biology

501

425

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Since their introduction, the interaction trap and other two-hybrid systems have been used to study protein-protein interactions. Despite their general use, little is known about the extent to which the degree of protein interaction determined by two-hybrid approaches parallels the degree of interaction determined by biochemical techniques. In this study, we used a set of lexAop-LEU2 and lexAop-lacZ reporters to calibrate the interaction trap. For the calibration, we used two sets of proteins, the Myc-Max-Mxi1 helix-loop-helix proteins, and wild-type and dimerization-defective versions of the lambda cI repressor. Our results indicate that the strength of interaction as predicted by the two-hybrid approach generally correlates with that determined in vitro, permitting discrimination of high-, intermediate-, and low-affinity interactions, but there was no single reporter for which the amount of gene expression linearly reflected affinity measured in vitro. However, some reporters showed thresholds and only responded to stronger interactions. Finally, some interactions were subject to directionality, and their apparent strength depended on the reporter used. Taken together, our results provide a cautionary framework for interpreting affinities from two-hybrid experiments.Biological systems depend on interactions between protein components. These interactions affect such diverse processes as the coordination of signal transduction by assembly of multisubunit complexes (57, 58), the regulation of apoptosis by the sequestration of Bax (54), and the control of gene expression through the selective association of transcription factors (19). Efforts to understand the functions of proteins often include identification and characterization of other cellular proteins with which they can interact. While some protein interactions are of high affinity and are easily detectable by physical techniques, a number of biologically important interactions, such as those of many enzymes with their substrates, are often relatively weak or transient and are not easily detectable by these methods.A number of approaches for studying protein association are in use, including cosedimentation through gradients, coimmunoprecipitation of purified proteins, assay of DNA binding activity for proteins that must dimerize to recognize a DNA site, and assay by two-hybrid systems (20) such as the interaction trap (31). In the last approach, a first protein (P1, or ''bait'') is fused to a known DNA-binding domain such as LexA (10) or GAL4 (41) and a second protein (P2) is fused to a transcriptional activation domain (AD). Coexpression of the two chimeric proteins in yeast cells in which the cognate binding site for the DNA-binding domain is located upstream of a reporter gene results in transcriptional activation of the reporter by the P2-fused AD if the chimeric proteins associate.Two-hybrid/interaction trap approaches have gained considerable popularity because they can detect novel interacting proteins that interact with a given bait by substituting...

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Expression of a cDNA derived from the yeast killer preprotoxin gene: implications for processing and immunity.

Cited by 45 publications

References 21 publications

Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

Topogenic Effect of Positively Charged N-terminal Amino Acid in ER Translocation of Yeast .ALPHA.-Factor Precursor.

Correlation of Two-Hybrid Affinity Data with In Vitro Measurements

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