Structural determinants in addition to the amino-terminal sorting sequence influence membrane localization of Escherichia coli lipoproteins

Abstract

The lipid-modified nine-residue amino-terminal sequence of the mature form of the major outer membrane lipoprotein of Escherichia coli contains information that is responsible for sorting to either the inner or outer membrane. Fusion of this sorting sequence to beta-lactamase is sufficient for localization of the resultant lipo-beta-lactamase to the outer membrane (J. Ghrayeb and M. Inouye, J. Biol. Chem. 259:463-467, 1984). Substitution of the serine adjacent to the amino-terminal lipid-modified cysteine residue of the sorting sequence with the negatively charged residue aspartate causes inner membrane localization (K. Yamaguchi, F. Yu, and M. Inouye, Cell 53:423-432, 1988). Fusion of the aspartate-containing nine-residue inner membrane localization signal to the normally outer membrane lipoprotein bacteriocin release protein does cause partial localization to the inner membrane. However, a single replacement of the glutamine adjacent to the amino-terminal lipid-modified cysteine residue of bacteriocin release protein with aspartate causes no inner membrane localization. Therefore, an aspartate residue itself lacks the information necessary for inner membrane sorting when removed from the structural context provided by the additional eight residues of the sorting sequence. Although the aspartate-containing inner membrane sorting sequence causes an almost quantitative localization to the inner membrane when fused to the otherwise soluble protein beta-lactamase, this sequence cannot prevent significant outer membrane localization when fused to proteins (bacteriocin release protein and OmpA) normally found in the outer membrane. Therefore, structural determinants in addition to the amino-terminal sorting sequence influence the membrane localization of lipoproteins.

Molecular characterization of a conserved 20-kilodalton membrane-associated lipoprotein antigen of Helicobacter pylori

“…Studies by Gennity et al (30) have subsequently shown, however, that structural features in addition to the aminoterminal sorting sequence influence the membrane localization of lipoproteins. Although most outer membrane lipoproteins had a serine at position +2, there were a number of exceptions to this rule (33), and most recently Poquet et al (55) have suggested, on the basis of studies with the aspartate +2-containing lipoprotein pullulanase, that there is no evidence that lipoproteins that cofractionate with the cytoplasmic membrane under certain conditions are actually associated with the membrane.…”

A gene at 59 minutes on the Escherichia coli chromosome encodes a lipoprotein with unusual amino acid repeat sequences

“…5). We (13,14). However, the presence of an aspartate residue in the second position after the modified cysteine residue can lead to a weak inner membrane sorting signal in a hybrid lipo-o-lactamase protein, and context-specific effects have also been previously noted (14).…”

Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2

“…Extracellular, periplasmic and cytoplasmic cell fractions of E. coli JM109 were prepared as described previously (Chang et al, 1993). The total membrane of E. coli was further separated into cytoplasmic and outer membrane fractions with a discontinuous sucrose gradient (Gennity et al, 1992) for unlabelled cells or with sodium lauryl sarcosinate (Filip et al, 1973) for [35S]methionine-labelled cultures. DNA sequencing and sequence analysis.…”