The dynamics of assembly of a cytoplasmic membrane protein in Escherichia coli.
The topology of integral cytoplasmic membrane proteins can be analyzed using alkaline phosphatase fusions by determining which constructs have low and which have high specific activity. We show that in all cases the enzymatic activity is due to the fraction of the alkaline phosphatase moiety of the fusion protein localized to the periplasm. We present evidence that these fusions can also be used to analyze the process of assembly of cytoplasmic proteins into the membrane. The rate of acquisition of protease resistance of the alkaline phosphatase moiety of such hybrid proteins is compared for fusions to periplasmic and cytoplasmic domains. We show that this process, which is assumed to be representative of export of alkaline phosphatase, is significantly slower for fusions to cytoplasmic and certain periplasmic domains than for most periplasmic domains. These results are discussed in the context of the normal assembly of integral membrane proteins.
Traxler
B
B
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Lee
C
C
Boyd
D
D
Beckwith
J
J
D018528:ATP-Binding Cassette Transporters; D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis* / Q000235:genetics; D010450:Endopeptidases / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005838:Genotype; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000096:biosynthesis*; D008954:Models, Biological; D008961:Models, Structural; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D015500:Chloramphenicol O-Acetyltransferase; D000469:Alkaline Phosphatase; D010450:Endopeptidases