GENETIC APPROACHES TO ANALYZING THE INSERTION & TOPOLOGY

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We will use a gene fusion approach to study the topological arrangement of integral membrane proteins. Fusions of alkaline phosphatase (AP) to periplasmic domains of such proteins give high AP enzymatic activity, while fusions of AP to cytoplasmic domains give low enzymatic activity. By isolating a large number of such fusions to the integral membrane proteins, MalF and leader peptidase, we hope to derive the topology of these membrane proteins. Fusions of betagalactosidase (betaGz) to periplasmic domains of membrane proteins exhibit low beta-Gz activity, while fusions of betaGz to cytoplasmic domains of the same protein give high activity - the converse of the situation with AP. Analysis of betaGz fusions to membrane proteins should also give information on topology. One betaGz fusion to MalF which has low betaGz activity, presumably due to its membrane insertion, will be used to obtain and characterize mutants which alter the ability of MalF to insert into the membrane. Selection of Lac+ derivatives of this strain should yield both mutations in the malF gene and unlinked mutants will be characterized and should yield information on 1) the signals in MalF responsible for its membrane insertion and 2) any cellular components that are involved in the insertion process.