In vivo expression of the lacY gene in two segments leads to functional lac permease

Journal Article · Fri Jun 01 00:00:00 EDT 1990 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.87.11.4325· OSTI ID:5393574

Bibi, E; Kaback, H R ^[1]

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1. Univ. of California, Los Angeles (USA)

The lacY gene of Escherichia coli was cut into two approximately equal-size fragments with Afl II and subcloned individually or together under separate lac operator/promoters in plasmid pT7-5. Under these conditions, lac permease is expressed in two portions: (i) the N-terminal portion (the N terminus, the first six putative transmembrane helices, and most of putative loop 7) and (ii) the C-terminal portion (the last six putative transmembrane helices and the C terminus). Cells harboring pT7-5 encoding both fragments transport lactose at about 30% the rate of cells expressing intact permease to a comparable steady-state level of accumulation. In contrast, cells expressing either half of the permease independently do not transport lactose. As judged by ({sup 35}S)methionine labeling and immunoblotting, intact permease in completely absent from the membrane of cells expressing lacY fragments either individually or together. Thus, transport activity must result from an association between independently synthesized pieces of lac permease. When the gene fragments are expressed individually, the N-terminal portion of the permease is observed inconsistently, and the C-terminal portion is not observed. When the gene fragments are expressed together, polypeptides identified as the N- and C-terminal moieties of the permease are found in the membrane. It is concluded that the N- or C-terminal halves of lac permease are proteolyzed when synthesized independently and that association between the two complementing polypeptides leads to a more stable, catalytically active complex.

• OSTI ID: 5393574
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Vol. 87:11; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
LABELLED COMPOUNDS
UPTAKE
MEMBRANE PROTEINS
BIOSYNTHESIS
MEMBRANE TRANSPORT
GENE REGULATION
CARBON 14 COMPOUNDS
CELL MEMBRANES
DNA-CLONING
ESCHERICHIA COLI
IODINE 125
LACTOSE
METHIONINE
POLYPEPTIDES
SULFUR 35
AMINO ACIDS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOHYDRATES
CARBON COMPOUNDS
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CLONING
DAYS LIVING RADIOISOTOPES
DISACCHARIDES
DNA HYBRIDIZATION
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
EVEN-ODD NUCLEI
HYBRIDIZATION
INTERMEDIATE MASS NUCLEI
INTERNAL CONVERSION RADIOISOTOPES
IODINE ISOTOPES
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
MEMBRANES
MICROORGANISMS
NUCLEI
ODD-EVEN NUCLEI
OLIGOSACCHARIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PEPTIDES
PROTEINS
RADIOISOTOPES
SACCHARIDES
SULFUR ISOTOPES
SYNTHESIS
550201* - Biochemistry- Tracer Techniques