Abstract
The in vivo membrane assembly of the mannitol permease, the mannitol Enzyme II (IImtl) of the Escherichia coli phosphotransferase system, has been studied employing molecular genetic approaches. Removal of the N-terminal amphiphilic leader of the permease and replacement with a short hydrophobic sequence resulted in an inactive protein unable to transport mannitol into the cell or catalyze either phosphoenol-pyruvate-dependent or mannitol 1-phosphate-dependent mannitol phosphorylation in vitro. The altered protein (68 kDa) was quantitatively cleaved by an endogenous protease to a membrane-associated 39-kDa fragment and a soluble 28-kDa fragment as revealed by Western blot analyses. Overproduction of the wild-type plasmid-encoded protein also led to cleavage, but repression of the synthesis of the plasmid-encoded enzyme by inclusion of glucose in the growth medium prevented cleavage. Several mtlA-phoA gene fusions encoding fused proteins with N-terminal regions derived from the mannitol permease and C-terminal regions derived from the mature portion of alkaline phosphatase were constructed. In the first fusion protein, F13, the N-terminal 13-aminoacyl residue amphiphilic leader sequence of the mannitol permease replaced the hydrophobic leader sequence of alkaline phosphatase. The resultant fusion protein was inefficiently translocated across the cytoplasmic membrane and became peripherally associated with both the inner and outer membranes, presumably via the noncleavable N-terminal amphiphilic sequence. The second fusion protein, F53, in which the N-terminal 53 residues of the mannitol permease were fused to alkaline phosphatase, was efficiently translocated across the cytoplasmic membrane and was largely found anchored to the inner membrane with the catalytic domain of alkaline phosphatase facing the periplasm. This 53-aminoacyl residue sequence included the amphiphilic leader sequence and a single hydrophobic, potentially transmembrane, segment. Analyses of other MtlA-PhoA fusion proteins led to the suggestion that internal amphiphilic segments may function to facilitate initiation of polypeptide trans-membrane translocation. The dependence of IImtl insertion on the N-terminal amphiphilic leader sequence was substantiated employing site-specific mutagenesis. The N-terminal sequence of the native permease is Met-Ser-Ser-Asp-Ile-Lys-Ile-Lys-Val-Gln-Ser-Phe-Gly.... The following point mutants were isolated, sequenced, and examined regarding the effects of the mutations on insertion of IImtl into the membrane: 1) S3P; 2) D4P; 3) D4L; 4) D4R; 5) D4H; 6) I5N; 7) K6P; and 8) K8P.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
The in vivo membrane assembly of the mannitol catedacrossthecytoplasmicmembraneandwas permease, the mannitol EnzymeI1 (IImt’o)f the Esche- largely found anchored to the inner membrane with richia coli phosphotransferase system, hasbeen stud- the catalytic domaionf alkaline phosphatase facing the ied employing molecular genetic approaches
A wild-type mtl genes under the control of their own promoter specific role for these sequences in the initial stagesof mem- was alsoconstructed andwas calledpMTL22 (Fig. 1).Expresbrane-protein association and insertion has been proposed sion of the mtlA and mtlD genes on this plasmid was not (Saier, 1989;Saier et al, 1989), and a tendency of correspoad- under the control of the lactose operator/promoter as indiing bacterial amphiphilic signal peptides to associate with cated by the inabilityof pFDX500 in trans tionfluence levels hydrophobic-hydrophilic interfaceshas been demonstrated of the enzymes in response t o isopropyl-1-thio-P-D-galacto
Since a 53-aminoacyl segment must carry line phosphatase activitiesof two mtlA-phoA fusion proteins more information than a 13-aminoacyl segment, we suggest were determined. Oneof these fusion proteins (F53) hasa 53- that the amphiphilic N-terminal segmentof IImt'may target residue segment of 1Pta1ttached to the mature pofaratlkaline the protein to theenvelope fraction of the cell and together phosphatase and exhibitshigh alkaline phosphatase activity, withthe hydrophobic segmentinitiateinsertionintothe while the other fusion protein (F13) has onlya13-residue cytoplasmic membrane
Summary
Dept. of Cancer Biology, Stanford University, Stanford, CA 94305. Lyon, F69622 Villeurbanne, France. N-terminal region shown in Fig. 1 was verified by both restriction enzyme mapping andnucleotide sequencing In this construction, synthesis of the fusion protein as well as the mannitol-1-phosphatedehydrogenase (encoded by the second gene in the mtl operon) was under the controlof the lactose operator/promoter. A wild-type mtl genes under the control of their own promoter specific role for these sequences in the initial stagesof mem- was alsoconstructed andwas calledpMTL22 (Fig. 1).Expresbrane-protein association and insertion has been proposed sion of the mtlA and mtlD genes on this plasmid was not (Saier, 1989;Saier et al, 1989), and a tendency of correspoad- under the control of the lactose operator/promoter as indiing bacterial amphiphilic signal peptides to associate with cated by the inabilityof pFDX500 in trans tionfluence levels hydrophobic-hydrophilic interfaceshas been demonstrated of the enzymes in response t o isopropyl-1-thio-P-D-galacto-. ThesesameHBlOl/pMTL22 cells grown inLB medium plus 0.2% mannitol were elongated as revealed by light microscopy in agreement with earlier reports (Lee and Saier, 1983a; Yamada et al, 1987) Ance has been attributed to inhibitioonf septation due to the overproduction of any oneof several integral membrane pro-
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