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Title: Sequential truncation of the lactose permease over a three-amino acid sequence near the carboxyl terminus leads to progressive loss of activity and stability

Abstract

Previous experiments are consistent with the notion that residues 396-401 (...SVFTLS...) at the carboxyl terminus of the last putative transmembrane helix of the lactose (lac) permease of Escherichia coli are important for protection against proteolytic degradation and suggest that this region of the permease may be necessary for proper folding. Stop codons (TAA) have now been substituted sequentially for amino acid codons 396-401 in the lacY gene, and the termination mutants were expressed from the plasmid pT7-5. With respect to transport, permease truncated at residue 396-or 397 is completely defective, while molecules truncated at residues 398, 399, 400, and 401, respectively, exhibit 15-25%, 30-40%, 40-45%, and 70-100% of wild-type activity. As judged by pulse-chase experiments with ({sup 35}S)methionine, wild-type permease or permease truncated at residue 401 is stable, while permease molecules truncated at position 400, 399, 398, 397, or 396 are degraded at increasingly rapid rates. The findings indicate that either the last turn of putative helix XII or the region immediately distal to helix XII is important for proper folding and protection against proteolytic degradation.

Authors:
; ; ;  [1]
  1. Univ. of California, Los Angeles (United States)
Publication Date:
OSTI Identifier:
5934351
Resource Type:
Journal Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America; (United States)
Additional Journal Information:
Journal Volume: 88:8; Journal ID: ISSN 0027-8424
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; LACTOSE; MEMBRANE TRANSPORT; TRANSFERASES; AMINO ACID SEQUENCE; CARBON 14 COMPOUNDS; ESCHERICHIA COLI; LABELLED COMPOUNDS; MEMBRANE PROTEINS; METHIONINE; SULFUR 35; TRACER TECHNIQUES; AMINO ACIDS; BACTERIA; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; CARBOHYDRATES; CARBON COMPOUNDS; CARBOXYLIC ACIDS; DAYS LIVING RADIOISOTOPES; DISACCHARIDES; DRUGS; ENZYMES; EVEN-ODD NUCLEI; ISOTOPE APPLICATIONS; ISOTOPES; LIGHT NUCLEI; LIPOTROPIC FACTORS; MICROORGANISMS; MOLECULAR STRUCTURE; NUCLEI; OLIGOSACCHARIDES; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; PROTEINS; RADIOISOTOPES; SACCHARIDES; SULFUR ISOTOPES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

McKenna, E, Hardy, D, Pastore, J C, and Kaback, H R. Sequential truncation of the lactose permease over a three-amino acid sequence near the carboxyl terminus leads to progressive loss of activity and stability. United States: N. p., 1991. Web. doi:10.1073/pnas.88.8.2969.
McKenna, E, Hardy, D, Pastore, J C, & Kaback, H R. Sequential truncation of the lactose permease over a three-amino acid sequence near the carboxyl terminus leads to progressive loss of activity and stability. United States. https://doi.org/10.1073/pnas.88.8.2969
McKenna, E, Hardy, D, Pastore, J C, and Kaback, H R. 1991. "Sequential truncation of the lactose permease over a three-amino acid sequence near the carboxyl terminus leads to progressive loss of activity and stability". United States. https://doi.org/10.1073/pnas.88.8.2969.
@article{osti_5934351,
title = {Sequential truncation of the lactose permease over a three-amino acid sequence near the carboxyl terminus leads to progressive loss of activity and stability},
author = {McKenna, E and Hardy, D and Pastore, J C and Kaback, H R},
abstractNote = {Previous experiments are consistent with the notion that residues 396-401 (...SVFTLS...) at the carboxyl terminus of the last putative transmembrane helix of the lactose (lac) permease of Escherichia coli are important for protection against proteolytic degradation and suggest that this region of the permease may be necessary for proper folding. Stop codons (TAA) have now been substituted sequentially for amino acid codons 396-401 in the lacY gene, and the termination mutants were expressed from the plasmid pT7-5. With respect to transport, permease truncated at residue 396-or 397 is completely defective, while molecules truncated at residues 398, 399, 400, and 401, respectively, exhibit 15-25%, 30-40%, 40-45%, and 70-100% of wild-type activity. As judged by pulse-chase experiments with ({sup 35}S)methionine, wild-type permease or permease truncated at residue 401 is stable, while permease molecules truncated at position 400, 399, 398, 397, or 396 are degraded at increasingly rapid rates. The findings indicate that either the last turn of putative helix XII or the region immediately distal to helix XII is important for proper folding and protection against proteolytic degradation.},
doi = {10.1073/pnas.88.8.2969},
url = {https://www.osti.gov/biblio/5934351}, journal = {Proceedings of the National Academy of Sciences of the United States of America; (United States)},
issn = {0027-8424},
number = ,
volume = 88:8,
place = {United States},
year = {Mon Apr 15 00:00:00 EDT 1991},
month = {Mon Apr 15 00:00:00 EDT 1991}
}