Mutant

About: Mutant is a research topic. Over the lifetime, 74520 publications have been published within this topic receiving 3477079 citations.

The Tomato R Gene Products I-2 and Mi-1 Are Functional ATP Binding Proteins with ATPase Activity

Abstract: Most plant disease resistance (R) genes known today encode proteins with a central nucleotide binding site (NBS) and a C-terminal Leu-rich repeat (LRR) domain. The NBS contains three ATP/GTP binding motifs known as the kinase-1a or P-loop, kinase-2, and kinase-3a motifs. In this article, we show that the NBS of R proteins forms a functional nucleotide binding pocket. The N-terminal halves of two tomato R proteins, I-2 conferring resistance to Fusarium oxysporum and Mi-1 conferring resistance to root-knot nematodes and potato aphids, were produced as glutathione S-transferase fusions in Escherichia coli. In a filter binding assay, purified I-2 was found to bind ATP rather than other nucleoside triphosphates. ATP binding appeared to be fully dependent on the presence of a divalent cation. A mutant I-2 protein containing a mutation in the P-loop showed a strongly reduced ATP binding capacity. Thin layer chromatography revealed that both I-2 and Mi-1 exerted ATPase activity. Based on the strong conservation of NBS domains in R proteins of the NBS-LRR class, we propose that they all are capable of binding and hydrolyzing ATP.

Endosomal transport function in yeast requires a novel AAA‐type ATPase, Vps4p

Abstract: In a late-Golgi compartment of the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y (CPY) are actively sorted away from the secretory pathway and transported to the vacuole via a pre-vacuolar, endosome-like intermediate. The vacuolar protein sorting (vps) mutant vps4 accumulates vacuolar, endocytic and late-Golgi markers in an aberrant multilamellar pre-vacuolar compartment. The VPS4 gene has been cloned and found to encode a 48 kDa protein which belongs to the protein family of AAA-type ATPases. The Vps4 protein was purified and shown to exhibit an N-ethylmaleimide-sensitive ATPase activity. A single amino acid change within the AAA motif of Vps4p yielded a protein that lacked ATPase activity and did not complement the protein sorting or morphological defects of the vps4 delta1 mutant. Indeed, when expressed at normal levels in wild-type cells, the mutant vps4 gene acted as a dominant-negative allele. The phenotypic characterization of a temperature-sensitive vps4 allele showed that the immediate consequence of loss of Vps4p function is a defect in vacuolar protein delivery. In this mutant, precursor CPY was not secreted but instead accumulated in an intracellular compartment, presumably the pre-vacuolar endosome. Electron microscopy revealed that upon temperature shift, exaggerated stacks of curved cisternal membranes (aberrant endosome) also accumulated in the vps4ts mutant. Based on these and other observations, we propose that Vps4p function is required for efficient transport out of the pre-vacuolar endosome.

A Heat-Inducible Transcription Factor, HsfA2, Is Required for Extension of Acquired Thermotolerance

Abstract: The expression of heat shock proteins (Hsps) induced by nonlethal heat treatment confers acquired thermotolerance (AT) toorganisms against subsequent challenges of otherwise lethal temperature. After the stress signal is removed, AT graduallydecays, with decreased Hsps during recovery. ATof sufficient duration is critical for sessile organisms such as plants to surviverepeated heat stress in their environment, but little is known regarding its regulation. To identify potential regulatorycomponents, we took a reverse genetics approach by screening for Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutantsthat show decreased thermotolerance after a long recovery (2 d) under nonstress conditions following an acclimation heattreatment. Among the tested mutants corresponding to 48 heat-induced genes, only the heat shock transcription factor HsfA2knockout mutant showed an obvious phenotype. Following pretreatment at 37 C, the mutant line was more sensitive to severeheat stress than the wild type after long but not short recovery periods, and this could be complemented by the introduction ofa wild-type copy of the HsfA2 gene. Quantitative hypocotyl elongation assay also revealed that AT decayed faster in theabsence of HsfA2. Significant reduction in the transcript levels of several highly heat-inducible genes was observed in HsfA2knockout plants after 4 h recovery or 2 h prolonged heat stress. Immunoblot analysis showed that Hsa32 and class I small Hspwere less abundant in the mutant than in the wild type after long recovery. Our results suggest that HsfA2 as a heat-inducibletransactivator sustains the expression of Hsp genes and extends the duration of AT in Arabidopsis.