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Journal of Bacteriology, April 2000, p. 2163-2169, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Net Charge of the First 18 Residues of the Mature Sequence Affects Protein Translocation across the Cytoplasmic Membrane of Gram-Negative Bacteria

Andrey V. Kajava,1,* Sergey N. Zolov,2 Andrey E. Kalinin,2,dagger and Marina A. Nesmeyanova2

Center for Molecular Modeling, CIT, National Institutes of Health, Bethesda, Maryland 20892,1 and Laboratory of Protein Secretion in Bacteria, Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia2

Received 22 October 1999/Accepted 21 January 2000

This statistical study shows that in proteins of gram-negative bacteria exported by the Sec-dependent pathway, the first 14 to 18 residues of the mature sequences have the highest deviation between the observed and expected net charge distributions. Moreover, almost all sequences have either neutral or negative net charge in this region. This rule is restricted to gram-negative bacteria, since neither eukaryotic nor gram-positive bacterial exported proteins have this charge bias. Subsequent experiments performed with a series of Escherichia coli alkaline phosphatase mutants confirmed that this charge bias is associated with protein translocation across the cytoplasmic membrane. Two consecutive basic residues inhibit translocation effectively when placed within the first 14 residues of the mature protein but not when placed in positions 19 and 20. The sensitivity to arginine partially reappeared again 30 residues away from the signal sequence. These data provide new insight into the mechanism of protein export in gram-negative bacteria and lead to practical recommendations for successful secretion of hybrid proteins.

* Corresponding author. Mailing address: Center for Molecular Modeling CIT, NIH, Bldg. 12A Room 2011, Bethesda, MD 20892. Phone: (301) 402-3043. Fax: (301) 402-2867. E-mail: kajava{at}helix.nih.gov.

dagger Present address: National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892.

Journal of Bacteriology, April 2000, p. 2163-2169, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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