Genetic studies on the inability of beta-galactosidase to be translocated across the Escherichia coli cytoplasmic membrane.
When a signal sequence is attached to beta-galactosidase, the normally cytoplasmic protein is unable to fully traverse the cytoplasmic membrane. We used a genetic approach to study those features of beta-galactosidase responsible for the block in translocation. By using both in vivo and in vitro techniques, fragments of beta-galactosidase were interposed between a signal sequence and alkaline phosphatase. The alkaline phosphatase acts as a sensor for any blocking effects of beta-galactosidase on export. From these studies, we show that multiple regions of beta-galactosidase contribute to its failure to be translocated. These results are most easily interpreted if the folding of beta-galactosidase or of domains of it is responsible for the block in export. In addition, in certain constructs, positively charged amino acids directly following the signal sequence interfered with export.
Lee
C
C
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Li
P
P
Inouye
H
H
Brickman
E R
ER
Beckwith
J
J
D000469:Alkaline Phosphatase / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005696:Galactosidases / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005838:Genotype; D008969:Molecular Sequence Data; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014161:Transduction, Genetic; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D005696:Galactosidases; D001616:beta-Galactosidase