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Characterization of a mutant form of SecA that alleviates a SecY defect at low temperature and shows a synthetic defect with SecY alteration at high temperature.
J Biochem 2000; 127(6):1071-9JB

Abstract

The secY205 mutant is cold-sensitive for protein export, with an in vitro defect in supporting ATP- and preprotein-dependent insertion of SecA into the membrane. We characterized SecA81 with a Gly516 to Asp substitution near the minor ATP-binding region, which suppresses the secY205 defect at low temperature and exhibits an allele-specific synthetic defect with the same SecY alteration at 42 degrees C. The overproduced SecA81 aggregated in vivo at temperatures above 37 degrees C. Purified SecA81 exhibited markedly enhanced intrinsic and membrane ATPase activities at 30 degrees C, while it was totally inactive at 42 degrees C. The trypsin digestion patterns indicated that SecA81 has some disorder in the central region of SecA, which encompasses residues 421-575. This conformational abnormality may result in unregulated ATPase at low temperature as well as the thermosensitivity of the mutant protein. In the presence of both proOmpA and the wild-type membrane vesicles, however, the thermosensitivity was alleviated, and SecA81 was able to catalyze significant levels of proOmpA-stimulated ATP hydrolysis as well as proOmpA translocation at 42 degrees C. While SecA81 was able to overcome the SecY205 defect at low temperature, the SecY205 membrane vesicles could not significantly support the translocation ATPase or the proOmpA translocation activity of SecA81 at 42 degrees C. The inactivated SecA81 molecules seemed to jam the translocase since it interfered with translocase functions at 42 degrees C. Based on these results, we propose that under preprotein-translocating conditions, the SecYEG channel can stabilize and activate SecA, and that this aspect is defective for the SecA81-SecY205 combination. The data also suggest that the conformation of the central region of SecA is important for the regulation of ATP hydrolysis and for the productive interaction of SecA with SecY.

Authors+Show Affiliations

Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10833277

Citation

Nakatogawa, H, et al. "Characterization of a Mutant Form of SecA That Alleviates a SecY Defect at Low Temperature and Shows a Synthetic Defect With SecY Alteration at High Temperature." Journal of Biochemistry, vol. 127, no. 6, 2000, pp. 1071-9.
Nakatogawa H, Mori H, Matsumoto G, et al. Characterization of a mutant form of SecA that alleviates a SecY defect at low temperature and shows a synthetic defect with SecY alteration at high temperature. J Biochem. 2000;127(6):1071-9.
Nakatogawa, H., Mori, H., Matsumoto, G., & Ito, K. (2000). Characterization of a mutant form of SecA that alleviates a SecY defect at low temperature and shows a synthetic defect with SecY alteration at high temperature. Journal of Biochemistry, 127(6), pp. 1071-9.
Nakatogawa H, et al. Characterization of a Mutant Form of SecA That Alleviates a SecY Defect at Low Temperature and Shows a Synthetic Defect With SecY Alteration at High Temperature. J Biochem. 2000;127(6):1071-9. PubMed PMID: 10833277.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of a mutant form of SecA that alleviates a SecY defect at low temperature and shows a synthetic defect with SecY alteration at high temperature. AU - Nakatogawa,H, AU - Mori,H, AU - Matsumoto,G, AU - Ito,K, PY - 2000/6/1/pubmed PY - 2000/9/23/medline PY - 2000/6/1/entrez SP - 1071 EP - 9 JF - Journal of biochemistry JO - J. Biochem. VL - 127 IS - 6 N2 - The secY205 mutant is cold-sensitive for protein export, with an in vitro defect in supporting ATP- and preprotein-dependent insertion of SecA into the membrane. We characterized SecA81 with a Gly516 to Asp substitution near the minor ATP-binding region, which suppresses the secY205 defect at low temperature and exhibits an allele-specific synthetic defect with the same SecY alteration at 42 degrees C. The overproduced SecA81 aggregated in vivo at temperatures above 37 degrees C. Purified SecA81 exhibited markedly enhanced intrinsic and membrane ATPase activities at 30 degrees C, while it was totally inactive at 42 degrees C. The trypsin digestion patterns indicated that SecA81 has some disorder in the central region of SecA, which encompasses residues 421-575. This conformational abnormality may result in unregulated ATPase at low temperature as well as the thermosensitivity of the mutant protein. In the presence of both proOmpA and the wild-type membrane vesicles, however, the thermosensitivity was alleviated, and SecA81 was able to catalyze significant levels of proOmpA-stimulated ATP hydrolysis as well as proOmpA translocation at 42 degrees C. While SecA81 was able to overcome the SecY205 defect at low temperature, the SecY205 membrane vesicles could not significantly support the translocation ATPase or the proOmpA translocation activity of SecA81 at 42 degrees C. The inactivated SecA81 molecules seemed to jam the translocase since it interfered with translocase functions at 42 degrees C. Based on these results, we propose that under preprotein-translocating conditions, the SecYEG channel can stabilize and activate SecA, and that this aspect is defective for the SecA81-SecY205 combination. The data also suggest that the conformation of the central region of SecA is important for the regulation of ATP hydrolysis and for the productive interaction of SecA with SecY. SN - 0021-924X UR - https://www.unboundmedicine.com/medline/citation/10833277/Characterization_of_a_mutant_form_of_SecA_that_alleviates_a_SecY_defect_at_low_temperature_and_shows_a_synthetic_defect_with_SecY_alteration_at_high_temperature_ L2 - https://joi.jlc.jst.go.jp/JST.Journalarchive/biochemistry1922/127.1071?lang=en&from=PubMed DB - PRIME DP - Unbound Medicine ER -