Abstract
This report describes an extensive mutational analysis of the most carboxyl-terminal membrane-spanning sequence of Escherichia coli lac permease (TM12). In addition to identifying residues important for lactose transport function, the analysis revealed that numerous mutations made lac permease highly toxic to cells. In the most extreme cases, production of such proteins at very low steady-state levels reduced cell viability greater than 10(4)-fold. Both frameshift and missense mutations led to toxicity, with the frameshift mutations having the strongest effects observed. The toxic missense mutations corresponded to changes in TM12 expected to interfere with membrane insertion or folding, such as the introduction of charged residues or prolines in the putative helix. The results suggest that cellular toxicity may be a relatively common consequence of mutations altering integral membrane protein folding. An analogous toxicity might contribute to the pathogenesis of several degenerative diseases caused by mutant membrane proteins, such as retinitis pigmentosa, Charcot-Marie-Tooth syndrome, and Alzheimer's disease.
Highlights
This report describes a mutational analysis of the 12th transmembrane segment (TM12)1 of Escherichia coli lac permease [5, 6]
Mutant Isolation—The sequence corresponding to the TM12 of lac permease (Fig. 1A) was mutagenized using an efficient “cassette” method (Fig. 1B) [9]
The lac permease gene gene was present in a small plasmid and carried a mutation creating a termination codon (UGA) corresponding to a site within TM12
Summary
This report describes a mutational analysis of the 12th transmembrane segment (TM12) of Escherichia coli lac permease [5, 6]. Our studies identify a set of TM12 residues that tolerate a variety of substitutions without loss of transport activity. These residues show an ␣-helical periodicity and may correspond to a side of the TM12 helix which faces the lipid bilayer. A number of missense and frameshift mutations were identified which render lac permease toxic to cells. The toxic missense changes did not cluster on either the tolerant or sensitive face of TM12 and corresponded to changes expected to cause misfolding of the mutant proteins
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