Mutant Membrane Protein Toxicity

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.Several diseases caused by mutant integral membrane proteins show dominant inheritance and are associated with tissue degeneration. These diseases include autosomal dominant retinitis pigmentosa, Charcot-Marie-Tooth disease, and Dejerine-Sottas syndrome (1-3). A simple hypothesis to help account for the pathogenesis of such diseases is that they reflect a general phenomenon in which mutations render membrane proteins directly toxic to cells (4).This report describes a mutational analysis of the 12th transmembrane segment (TM12) 1 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 also 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. MATERIALS AND METHODSBacteria and Plasmids-The strains and plasmids used in this study are listed in Table I. Plasmid pCS112 is a pBR322-based plasmid derived from pCM472 (7) which carries a modified lacY with a UGA nonsense codon corresponding to codon 386 and a StyI restriction site at the NH 2 -terminal end of the sequence encoding TM12 (Table I). To construct pCS112, a SspI-SspI fragment that included phoA was first deleted from pCM472. Second, the StyI site in the tet gene was removed by StyI cleavage, followed by DNA polymerase I Klenow fragment and T4 DNA ligase treatments. Third, a StyI cleavage site was introduced at a position corresponding to the NH 2 -terminal end of TM12, and a UGA termination codon (codon 386) and SpeI cleavage site were introduced within the sequence corresponding to TM12 by site-directed muta...

Threonine-978 in the Transmembrane Segment of the Multidrug Efflux Pump AcrB of Escherichia coli Is Crucial for Drug Transport as a Probable Component of the Proton Relay Network

“…In the lactose permease LacY, Thr348, located near the end of TM helix XI, was identified as being required for transport activity by cysteine scanning mutagenesis (5), and Thr393 in TM helix XII was identified as essential by random mutagenesis of the residues in this helix (20). However, the crystal structure (1) shows that these residues are far away from the proton relay region, and the roles played by these Thr residues are not clear at present.…”

Evidence for a dynamic role for proline376 in the purine–cytosine permease of Saccharomyces cerevisiae

“…Owing to the probable instability of the DNA sequence target of the desired mutations and because of the likely toxicity of certain mutant forms of the protein in bacteria, all the desired mutants were not obtained. Missense mutations have already been shown to render lac permease toxic to E. coli cells [36]. This toxicity is assumed to be a relatively common consequence of mutations that alter integral membrane protein folding.…”

Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter

“…ENTs are similar to MFS members in their predicted membrane topology accommodating a large loop between TMs 6 and 7, although they are conjectured to have 11 TMs with an extracellular COOH terminus (34). Although the exclusion of TM12 in the ENT prediction from the MFS template is a concern, an extensive mutagenesis study of TM12 of the lactose permease, an MFS member, indicates that residues in this domain do not participate in ligand binding or translocation (35). The validity of the predicted helix packing in the ENT model (Fig.…”