Many membrane proteins exist and function as oligomers, but how monomers oligomerize in the cell membrane remains poorly understood. AcrB is an obligate homo-trimer. We previously found that the folding of individual subunit precedes oligomerization. Following folding, individual AcrB subunits must locate and interact with each other in order to dimerize and eventually trimerize. It has been unclear if AcrB trimerization is a spontaneous process following the chance encounter and random assembling mechanism. In other words, it is currently unknown whether monomeric subunits diffuse freely to search for each other after they are co-translationally inserted and folded into the cell membrane. Using four sets of experiments exploiting AcrB variants with different fusion tags, disulfide trapping, and activity measurement, here we showed that AcrB variants co-expressed in the same Escherichia coli cell did co-assemble into hybrid trimers in vivo. However, the level of co-assembly measured experimentally was not consistent with calculations derived from random assembling. The potential role of the polysome structure during protein translation and the resultant clustering effect were discussed as a potential explanation for the observed bias in AcrB subunit assembling in vivo. Our results provide new insights into the dynamic assembling and equilibration process of obligate homo-oligomeric membrane proteins in the cell membrane.
|Number of pages||12|
|Journal||Journal of Molecular Biology|
|State||Published - Oct 12 2012|
Bibliographical noteFunding Information:
We acknowledge the University of Kentucky Proteomics Core that is partially supported by grants from the National Center for Research Resources ( 5P20RR020171-09 ). We thank the National Science Foundation ( MCB 1158036 ) (Y.W.), Kentucky NASA EPSCoR ( NNX10AV39A ) (Y.W.), Oak Ridge Associated Universities Ralph E. Powe Junior Faculty Enhancement Award (Y.W.), and the National Institute of General Medical Sciences ( 8 P20 GM103486-09 ) (H.Z.) for supporting this work.
- membrane protein oligomerization
- obligate oligomer
- protein folding and assembling
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology