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Structure/function relationships in the Escherichia coli mannitol permease: identification of regions important for membrane insertion, substrate binding and oligomerization.

Abstract：

:The Escherichia coli mannitol permease (EIIMtl) of the phosphoenolpyruvate-dependent phosphotransferase system is a 68-kDa membrane protein that carries out the concomitant transport and phosphorylation of D-mannitol. Previous studies indicated that there are ca. 6 membrane-spanning helices within the N-terminal half of the protein, while the hydrophilic C-terminal half was shown to be exposed in the cytoplasm. In the present study, an analysis of C-terminally truncated EIIMtl mutants showed that proteins from which only the cytoplasmic domain has been deleted were present in the membrane at > or = 50% the amount of the intact protein. However, deletion proteins smaller than ca. 34 kDa were present in the membrane at only about 20% the amount of the intact protein. We also constructed a plasmid that encodes the first 43 amino acid residues of ELLMtl fused to residues 378 to 637 (the C-terminal domain). The corresponding protein was associated with the cytoplasmic membrane. These results show that the first 43 amino acid residues of the N terminus are sufficient for membrane localization, although the region comprising the last 2 membrane-spanning helices appears to be important for maximum stability and/or efficient membrane insertion of the complete N-terminal domain. Further studies of these deletion proteins showed that binding of mannitol to the permease occurs even if the entire cytoplasmic domain is absent, but is abolished if the last putative membrane-spanning region is removed. Finally, regions of the protein within the membrane-bound domain were identified that influence the oligomerization state of the protein. These results further define domains of this multifunctional transport protein that are important for membrane insertion, stability, substrate binding and oligomerization.

journal_name

Res Microbiol

journal_title

Research in microbiology

authors

Briggs CE,Khandekar SS,Jacobson GR

doi

10.1016/0923-2508(92)90003-7

subject

Has Abstract

pub_date

1992-02-01 00:00:00

pages

139-49

issue

eissn

0923-2508

issn

1769-7123

pii

0923-2508(92)90003-7

journal_volume

143

pub_type

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Structure/function relationships in the Escherichia coli mannitol permease: identification of regions important for membrane insertion, substrate binding and oligomerization.