Targeting of fusion proteins to organelles in yeast and tobacco: investigation of the molecular basis of toxin susceptibility in mitochondria of maize (Zea mays L.) with the Texas male-sterile cytoplasm
Date of Award
Doctor of Philosophy
The T-URF13 protein from mitochondria of maize with the Texas male-sterile (cms-T) cytoplasm is implicated in the causation of cytoplasmic male sterility and sensitivity to host-specific fungal toxins produced by Helminthosporium maydis race T (HmT toxin) and Phyllosticta maydis (Pm toxin), as well as to the insecticide methomyl. Whether T-URF13 is responsible for toxin sensitivity in cms-T mitochondria was addressed. A chimeric gene coding for T-URF13 fused to the mitochondrial leader peptide from the Neurospora crassa ATP synthase subunit 9 precursor was expressed in the yeast Saccharomyces cerevisiae. The fusion protein encoded by this gene was translocated into the membrane fraction of mitochondria and processed. HmT and Pm toxins and methomyl inhibited growth of yeast cells expressing the gene fusion on medium containing glycerol as sole carbon source and stimulated NADH-supported respiration by isolated mitochondria from these cells. These effects were not observed in yeast cells expressing a non-mitochondrial form of T-URF13. The results indicate that T-URF13 is the causative agent of toxin and methomyl sensitivity in T maize, and suggest that mitochondrial localization of T-URF13 is critical for its function;In a study to identify yeast mitochondrial leader peptides that can direct protein transport to mitochondria of tobacco (Nicotiana tabacum), a mitochondrial leader peptide from cytochrome oxidase subunit V[subscript]a of yeast (S. cerevisiae) was found to function as protein targeting signal with dual specificity in transgenic tobacco; this leader peptide directed the import of chloramphenicol acetyltransferase into both mitochondria and chloroplasts. The form of the protein in the two organelles was similar in size to the protein inside yeast mitochondria, suggesting a conserved mechanism of processing. These findings provide a potential system for studying organelle specificity of protein targeting in plants;Chimeric genes coding for T-URF13 were introduced into tobacco in order to examine any effect of T-URF13 on pollen fertility. Transgenic tobacco was not male-sterile nor sensitive to the toxins. The apparent absence of gene products in the transformants most likely explains the absence of these phenotypes.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Huang, Jintai, "Targeting of fusion proteins to organelles in yeast and tobacco: investigation of the molecular basis of toxin susceptibility in mitochondria of maize (Zea mays L.) with the Texas male-sterile cytoplasm " (1990). Retrospective Theses and Dissertations. 9374.