Abstract
SecG, an integral membrane component of the Escherichia coli preprotein translocase, contributes to the efficiency of the export process by undergoing cycles of topology inversion in the membrane, coupled with the insertion-deinsertion cycles of SecA. We have previously identified sec alleles of secG that cause a generalized secretion defect. In this study, by screening mutagenized secG libraries for suppressors of a malE signal sequence mutation, we isolated prl alleles of secG. By analogy with secY/prlA, secA/prlD, and secE/prlG, secG could therefore be called secG/prlH. The prlH mutations affect 13 codons distributed along the secG sequence, and none map to the codons affected by sec mutations. prlH suppressors suppress a variety of signal sequence mutations and they allow export of alkaline phosphatase lacking its entire signal sequence. Although secG was not identified in previous selections for prl mutants, several prlH alleles are as strong as the strongest known prlG alleles of secE. Some prlH alleles can also promote the export of alkaline phosphatase fused to predicted cytoplasmic domains of UhpT, an integral membrane protein. These results support the notion that SecG contributes to signal sequence recognition, and suggest that it may also contribute to the topology of integral membrane proteins.
Highlights
Mic and outer membrane proteins completely devoid of signal sequences are exported quite efficiently in certain prl suppressor strains [15, 16]
Isolation of prl Alleles of secG—We have used as a reporter a well characterized periplasmic protein of E. coli, MBP, whose export is required for maltose uptake [46]
Signal sequence mutations in the malE gene cause the accumulation of the precursor form of MBP in the cytoplasm and affect the ability of E. coli to use maltose as a carbon source [38]
Summary
They were constructed by P1mediated transduction or transformation [40]. The plasmid encoding prlHϩ (pHϩ) is identical to psecGϩ, a pACYC184 derivative described previously [32]. Plasmids encoding prlH mutants (pH1 to pH6) are. Strains MC1000 DHB3 SB80 SB81 Mph Mph Mph Mph MC4100 MM2 MM3 MM4 SB77 SB78 SB70. Plasmids pACYC184 pHϩ pH1 pH2 pH3 pH4 pH5 pH6 prl Mutations in the E. coli secG Gene
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