Dihydrofolate reductase (mouse) and beta-galactosidase (Escherichia coli) can be translocated across the plasma membrane of E. coli.
Hybrid genes were constructed. One, ompA153-dfr, encoded the precursor of the 325 residue Escherichia coli outer membrane protein OmpA up to residue 153 which was fused to the complete 186-residue dihydrofolate reductase of the mouse. The other, ompA219-lacZ, coded for the same precursor up to residue 219 which was fused to 1017 COOH-terminal residues of the 1023-residue subunit of the beta-galactosidase of E. coli. Full expression of the ompA153-dfr gene caused accumulation of its precursor and of that of the chromosomally encoded OmpA protein. When the amount of product was reduced, no pro-OmpA and very little pro-hybrid protein accumulated. The precursor was processed and the mature protein was fully accessible to trypsin in permeabilized cells. Expression of the ompA219-lacZ gene led to the presence of the hybrid protein at only 20-30% of the amount expected. About 20% of it appeared to be incorporated in the outer membrane. All of the hybrid was quantitatively accessible to trypsin in permeabilized cells. When the hybrid gene was overexpressed, the protein was found associated with the plasma membrane in the cytosol. It is concluded that both beta-galactosidase and dihydrofolate reductase could quantitatively traverse the plasma membrane, provided the amounts synthesized were sufficiently small.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology*; D005696:Galactosidases / Q000378:metabolism*; D005786:Gene Expression Regulation; D051379:Mice; D008854:Microscopy, Electron; D009943:Organophosphorus Compounds; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014295:Trimethoprim / Q000494:pharmacology; D001616:beta-Galactosidase / Q000378:metabolism*
D009943:Organophosphorus Compounds; C008705:octamethyl pyrophosphoramide; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D005696:Galactosidases; D001616:beta-Galactosidase