Residues flanking the COOH-terminal C-region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase and the extent of coupling of its co-translational translocation and proteolytic processing in vitro.

S F Nothwehr,S D Hoeltzli,K L Allen,M O Lively,J I Gordon

doi:10.1016/s0021-9258(18)45810-3

S F Nothwehr, S D Hoeltzli + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(18)45810-3

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Abstract

The polar, COOH-terminal c-region of signal peptides has been considered to be most important for influencing the efficiency and fidelity of signal peptidase cleavage while the hydrophobic core or h-region appears indispensable for initiating translocation. To identify structural features of residues flanking the c-region that influence the fidelity and efficiency of signal peptidase cleavage as well as co-translational translocation, we introduced six amino acid substitutions into the COOH terminus of the hydrophobic core and seven substitutions at the NH2 terminus of the mature region (the +1 position) of a model eukaryotic preprotein-human pre(delta pro)apoA-II. This preprotein contains several potential sites for signal peptidase cleavage. The functional consequences of these mutations were assayed using an in vitro co-translational translocation/processing system and by post-translational cleavage with purified, detergent-solubilized, hen oviduct signal peptidase. The efficiency of translocation could be correlated with the hydrophobic character of the residue introduced at the COOH terminus of the h-region. Some h/c boundary mutants underwent co-translational translocation across the microsomal membrane with only minimal cleavage yet they were cleaved post-translationally by hen oviduct signal peptidase more efficiently than other mutants which exhibited a high degree of coupling of co-translational translocation and cleavage. These data suggest that features at the COOH terminus of the h-domain can influence "presentation" of the cleavage site to signal peptidase. The +1 residue substitutions had minor effects on the extent of co-translational translocation and processing. However, these +1, as well as h/c boundary mutations, had dramatic effects on the site of cleavage chosen by signal peptidase, indicating that residues flanking the c-region of this prototypic eukaryotic signal peptide can affect the fidelity of its proteolytic processing. The site(s) selected by canine microsomal and purified hen oviduct signal peptidase were very similar, suggesting that "intrinsic" structural features of this prepeptide can influence the selectivity of eukaryotic signal peptidase cleavage, independent of the microsomal membrane and associated translocation apparatus.

Highlights

The polar, COOH-terminal c-region of signal peptides has been considered to be most important for influencing the efficiency and fidelity of signal peptidase cleavage while the hydrophobic core or h-region appears indispensable for initiating translocation
Some h/c boundary mutants underwent co-translational translocation across the microsomal membrane with only minimal cleavage yet they were cleaved post-translationally by hen oviduct signal peptidase more efficiently than other mutants which exhibited a high degree of coupling of co-translational translocation and cleavage
The + 1 residue substitutions had minor effects on the extent of co-translational translocation and processing. These +l, as well as h/c boundary mutations, had dramatic effects on the site of cleavage chosen by signal peptidase, indicating that residues flanking the c-region of this prototypic eukaryotic signal peptide can affect the fidelity of its proteolytic processing

Summary

Features Influencing Cleavage of Eukaryotic Signal Peptides

At the -1 and -3 positions [9, 14]. Systematic site-directed mutagenesis of both eukaryotic [15, 16] and prokaryotic preproteins [17,18] have confirmed the importance of the physical-chemical properties of the -1 residue in establishing a proper “context” for signal peptidase cleavage. Previous work from our laboratory has shown that at least two sites of cleavage in a model preprotein, human pre(Apro)apolipoprotein(apo)A-II, can “compete” for recognition by signal peptidase [15] This feature allowed us to use site-directed mutagenesis to manipulate the location of the h/c boundary and determine that in addition to the importance of features at the -1 and -3 positions, the processing machinery tends to conserve a distance of 4-5 residues from its h/c junction to the site of cleavage [19]. Data from experiments which analyze the extent of coupling of translocation and cleavage, and the efficiency of post-translational cleavage by detergent-solubilized HOSP, suggest that the physical-chemical properties of amino acids located in both positions are important for proper presentation of the cleavage site to signal peptidase

RESULTS AND DISCUSSION

Amino Acid Position

The supernatant fractions from each treatment were loaded in lanes

TABLE I