Kinetic analysis of lamB mutants suggests the signal sequence plays multiple roles in protein export.

J Stader,S A Benson,T J Silhavy

doi:10.1016/s0021-9258(18)66832-2

Abstract

We have developed a quantitative assay to measure the rate of processing of precursor LamB into mature protein and have used this assay to characterize 10 previously isolated and 3 new lamB signal sequence mutants. The data suggest that the LamB signal sequence serves a complex function. Our assay has revealed five types of signal sequence defect: 1) a strong kinetic defect resulting from alteration of the secondary structure in the putative alpha-helical region in the hydrophobic core, 2) a strong, or 3) a weak kinetic defect due to placement of a charged residue in the hydrophobic core, 4) decreased synthesis of LamB, and 5) both a decrease in synthesis and a strong kinetic defect. The effect of an extragenic suppressor, prlA4 on the rate of processing pLamB containing signal sequence mutations was also examined and compared to the rates in wild-type strains. It was found that prlA4 increases the rate of processing in some, but not all, mutants having a kinetic defect while having no effect on the decreased synthesis seen in mutants of types 4 and 5.

Highlights

We have developed a quantitative assay to measure mutations in certain genetic loci have been shown to have a the rate of processingof precursorLamB into mature pleiotropic effect on protein export
Most N-terminal molecular function(s1performed by this export signal we have signal sequences range in size from 13 to 36 amino acids and devised a quantitative assay and used it to analyze the effects have similar characteristics beginning with a hydrophilic N of a large collection of LamB signal sequence mutations
This terminus of about 2 to 5 polar amino acids followed by 1or 2 collection includes all of the mutations previously isolated basic residues, a hydrophobic core of 10 to 15 amino acids, (Emr et al, 1978; Emr and Silhavy, 1980, 1983; Hall et al, and a shortregion of 5 or 6more polar residues serving as the 1982a,1983; Schwartz et al, 1981) plus 3 new mutations signal peptidase cleavage site

Summary

MATERIALS ANDMETHODS

Homologies donot seem to exist among the signal Bacterial Strains-All strains are derivatives of the E. coliK12 sequences of secreted proteins at thenucleotide or amino acid strain MC4100 (F- araD139 flbB5301 A(lac)U169 rpsLI50 ptsF25 levels, it has been found that (i) signal sequences are inter- deoCl reUl thi (Casadaban, 1976)). Homologies donot seem to exist among the signal Bacterial Strains-All strains are derivatives of the E. The nomenclature used in this changeable between exported proteins(Palva et al, 1982; Takahara et al, 1985; Talmadge et aL, 1980; Tommassen et al, 1983) and (ii) certain signal sequences are functional in heterologous systems (Roggenkamp et al, 1981; Talmadge et al, 1980). Signal sequence mutations in strains SE2069-99were isolated as MalRrevertants (SE2069, 70, 71, 73, 78, and 99 (Emr and Silhavy, 1980)) or Lac+ revertants (SE2096 (Emr and Silhavy, 1982))of lac fusion strain pop3186 (MC4100@ (lam€!lacZ)hyb (Silhavy et al, 1977)). Three new signal sequence mutationst,hosein strains ECB225 (MC4100 lamBlIO), ECB308 (MC4100lamB1251, and ECB369 (MC4100 lamBI6U),were isolated richia coli, this contention is based largely on the fact that by selecting Lac+coloniesin the lac fusion strain pop3299 The effect of the Analysis of LamB Signal Sequence Mutations (r2)

Reference lumB mutation

RESULTS

DISCUSSION