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Title:Genetic dissection of proline transport by Salmonella typhimurium
Author(s):Myers, Richard Stanley
Doctoral Committee Chair(s):Maloy, Stanley R.
Department / Program:Biology, Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Microbiology
Abstract:Transport is an essential and often rate-limiting step in the growth and metabolism of cells. However, very little is known about the molecular mechanism of substrate transport. Proline transport by Salmonella typhimurium is a good model system for studying the molecular mechanism of ion-driven transport systems. The putP gene encodes proline permease, a sodium/proline symport system. In order to identify the active site of proline permease, we isolated a large number of rare putP missense mutations that alter the kinetics and specificity of transport.
Mutations that affect the substrate binding site of proline permease were isolated by screening for rare missense mutants that fail to transport some, but not all of the normal substrates. By localized mutagenesis, seven different phenotypic classes of substrate specificity mutants were isolated. The mutations cluster in three small deletion intervals of the putP gene. Kinetic analysis of proline transport identified two classes of kinetic defects: one class had a much greater Km than the wild type, and the other class had a much lower Vmax than the wild type. The properties of the Km mutants suggest they affect amino acids required for substrate binding whereas the Vmax mutants might affect substrate translocation.
In order to identify the cation binding site of proline permease, I isolated a large number of putP mutations that alter the cation specificity of proline permease. Localized mutagenesis of the putP gene yielded two phenotypic classes of cation specificity mutants. One class of mutants appears to be defective in Li$\sp+$ binding, while the other class may be defective in both Li$\sp+$ and Na$\sp+$ binding. The mutations cluster in two small deletion intervals of the putP structural gene. The map location of the cation specificity mutations is different than the location of substrate specificity mutations, suggesting that the active site of proline permease is composed of two domains: a substrate binding domain and a cation binding domain.
In order to facilitate physical characterization of proline permease, a large number of putP-lacZ gene fusions were isolated and characterized. A representative proline permease hybrid protein was purified. The gene fusions should be useful for probing the membrane topology of proline permease. The fusion proteins may be useful for isolating antibody directed against proline permease.
Issue Date:1989
Rights Information:Copyright 1989 Myers, Richard Stanley
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI8924910
OCLC Identifier:(UMI)AAI8924910

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