Modification and processing of internalized signal sequences of prolipoprotein in Escherichia coli and in Bacillus subtilis.

S Hayashi,S Chang,S Y Chang,C Z Giam,H C Wu

doi:10.1016/s0021-9258(18)89086-x

Abstract

We have cloned the Escherichia coli lipoprotein structural gene (lpp) into a shuttle vector and studied its expression in both E. coli and in Bacillus subtilis. Using in vitro gene fusion techniques, the lpp gene was placed under the control of the promoter for the erythromycin-resistance (ery) gene. This fusion gene directed the synthesis of Braun's prolipoprotein which can be subsequently processed into the mature lipoprotein. In addition to the prolipoprotein, two ery-lpp hybrid proteins containing a 45- and a 22-amino acid extension preceding the NH2 terminus of prolipoprotein, respectively, are also synthesized in E. coli. The synthesis of these three proteins appears to involve the utilization of three distinct translation initiation sites. In B. subtilis, only two proteins are synthesized, the hybrid protein with a 45-amino acid extension and the prolipoprotein. In both E. coli and B. subtilis, the precursor forms of the hybrid proteins are lipid-modified, and they are processed to mature lipoprotein in vivo. These results indicate that internalized signal sequence containing the prolipoprotein modification and processing site (Leu-Ala-Glys-Cys) can function normally and permit the modification of hybrid proteins to lipid-modified precursors which can be subsequently processed by the globomycin-sensitive prolipoprotein signal peptidase.

Highlights

From the Department of Microbiology, UniformedServices University of the Health Sciences, Bethesda, Maryland 20814-4799 and the $.Department of Microbial Genetics, CetusCorporation, Emeryville, California94608
In B. subtilis, only two proteins are synthesized, the hybrid protein with a 45-amino acid extension and the prolipoprotein. In both E. coli and B . subtilis, the precursor forms of the hybrid proteins are lipid-modified, and they are processed to mature lipoprotein in vivo. These results indicate that internalized signal sequence containing the prolipoprotein modification and processing site (Leu-Ala-Glys-Cys) can function normally and permit the modification of hybrid proteins to lipidmodified precursors which can be subsequently processed by the globomycin-sensitive prolipoprotein signal peptidase
While the biosynthesis of membrane-bound and lipid-modified penicillinase appears to involve similar modification and processing enzymes, as evidenced by the specific inhibition of the processing of modified prepenicillinase by globomycin in both organisms, the rateof maturation of prepenicillinase appears to be much slower in B. subtilis than that in E. coli

Summary

THE JOURNALOF BIOLOGICACLHEMISTRY

In addition to the prolipoprotein, two ery-lpp hybrid proteins containing a 45- and a 22-amino acid extension preceding the NH2 terminus of prolipoprotein, respectively, are synthesized in E. coli. Erythromycin (0.05 pg/ml final concentration) was added to B. subtilis cells harboring plasmid pSYC820 in order to induce the expression of the cloned lpp gene. Determination of the NH2-Terminal Sequences in Prolipoprotein and Lipoprotein by Edman Degradation of Labeled Proteins-E. coli cells harboring plasmid pSYC820were labeled with [3H]lysine (1 mCi/5 ml) and [35S]methionine(100 pCi/5 ml) for 10 min in the coding sequence of lpp was isolated. Plasmid pBD15 (which isidenticalto pE194cop-6 mutant, see Ref. 7 ) DNA was digested with MboI and HaeIII, and the310-base pair MboIHue111 fragment containing the promoter and the NH,-terminal portion of the erythromycin-resistance (ery)gene was presence of globomycin (30 pg/ml).

Ser Ser

SEQUENCE CYCLE

Findings

DISCUSSION