Abstract
We used the maltose transport complex MalFGK2 of the Escherichia coli cytoplasmic membrane as a model for the study of the assembly of hetero-oligomeric membrane protein complexes. Analysis of other membrane protein complexes has led to a general model in which a unique, ordered pathway is followed from subunit monomers to a final oligomeric structure. In contrast, the studies reported here point to a fundamentally different mode for assembly of this transporter. Using co-immunoprecipitation and quantification of interacting partners, we found that all subunits of the maltose transport complex efficiently form heteromeric complexes in vivo. The pairwise complexes were stable over time, suggesting that they all represent assembly intermediates for the final MalFGK2 transporter. These results indicate that several paths can lead to assembly of this oligomer. We also characterized MalF and MalG mutants that caused reduced association between some or all of the subunits of the complex with this assay. The mutant analysis highlights some important motifs for subunit contacts and suggests that the promiscuous interactions between these Mal proteins contribute to the efficiency of complex assembly. The behaviors of the wild type and mutant proteins in the co-immunoprecipitations support a model of multiple assembly pathways for this complex.
Highlights
We study the assembly of the maltose transport complex, a simple hetero-oligomeric membrane protein complex in the cytoplasmic membrane of Escherichia coli [6, 7] (Fig. 1)
The Effect of Insertion Mutants of MalF and MalG on Pairwise Association of the Mal Proteins—Previously, we isolated a number of malF and malG derivatives [17]1 that each expresses a mutant protein containing a similar insertion of 31 residues at different locations in the protein. We examined these for their ability to transport maltose and to tetramerize into a MalFGK2 complex and identified several that are transport-deficient and apparently have defects in the oligomerization of the maltose transport complex [17, 21]
Because of the quantity of each pairwise complex and the similar stability of these complexes in pulse-chase analyses, we propose that all of these heteromeric species are intermediates that can participate in the assembly of the maltose transport complex
Summary
Bacterial Strains and Plasmids—Various compatible plasmids (Table I) were combined in strain BT6 (MC1000 ⌬malB101 zjb::Tn5 (⌬malE,F,G,K,lamB,malM) F’128lacIq). For experiments with plasmid-borne mal alleles, 1 ml of culture was induced with 1 mM isopropyl-1-thio--D-galactopyranoside for 5 min and labeled by the addition of 79 or 158 Ci [35S]Met (Expre35S35S label, PerkinElmer Life Sciences) for 5 min. Labeling was stopped by the addition of 0.05% cold Met, and the cultures were immediately placed on ice. For pulse-chase experiments, 2 ml of culture were induced with 1 mM isopropyl-1-thio--D-galactopyranoside for 5 min, and 158 Ci of [35S]Met was added. After 15 min of additional incubation at 37 °C, a 1-ml chase sample was placed on ice. The cells were harvested, washed one time with 50 mM Tris-HCl, pH 8.0, 1 mM EDTA (buffer A), and resuspended in 0.5 ml of buffer A. For the in vitro mixing studies, the lysates of cells expressing different combinations of Mal subunits were solubilized with dodecyl maltoside as above and mixed. Differences in association between Mal subunits of Ϯ0.02– 0.03 are not considered significant in our analysis, consistent with the background present in control studies between MalF and TsrR47 (see “Results” below)
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