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Eukaryotic signal peptide structure/function relationships. Identification of conformational features which influence the site and efficiency of co-translational proteolytic processing by site-directed mutagenesis of human pre(delta pro)apolipoprotein A-II


Eukaryotic signal peptide structure/function relationships. Identification of conformational features which influence the site and efficiency of co-translational proteolytic processing by site-directed mutagenesis of human pre(delta pro)apolipoprotein A-II



Journal of Biological Chemistry 264(7): 3979-3987



ISSN/ISBN: 0021-9258

PMID: 2537299

The structural features with influence the efficiency and site of clevage by eukaryotic signal peptidase have not been fully defined. Human pre (.DELTA. pro) apoliprotein A-II is a useful model system for such an analysis. We have recently shown that a panel of mutants which have amino acids of varying physical-chemical properties substituted for the -1 residue (Ala20) of its signal peptide exhibit cleavage at one of three potential sites or, in some cases, bidirected cleavage (Folz, R. J., Nothwehr, S. F., and Gordon, J. I. (1988) J. Biol. Chem. 263, 2070-2078). In this present study, a subset of the pre (.DELTA. pro) Xaa20 apoA-II mutants were used to identify conformational features, located COOH-terminal to the hydrophobic core domain, which regulate its cotranslational translocation and proteolytic processing. Proline residues were substituted at postions 13, 14 or 15 in ten of the original position 20 mutant preproteins in order to induce formation of a .beta.-turn structure at various positions upstream of the cleavage site. The effects of these mutations were assessed using an in vitro transcription/translation/microsome processing assay. Substitution of proline at position 13 resulted in a dramatic decrease in processing efficiency in all Xaa20 sequence contexts, while processing progressively increased when the proline was moved "downstream" to positions 14 and 15. Elongating the hydrophobic core of the Pro13 mutants by 1 residue reversed this effect. NH2-terminal sequence analysis of 34 co-translationally processed mutants revealed that the optimum distance between the site of the proline introduction and the site of co-translational cleavage was 4-5 residues. However, this distance could be altered by the presence of certain amino acids at positions -1 and -3. The data suggest that several structural motifs NH2-terminal to residues -1 and -3 influence the location, site, and efficiency of cleavage by eukaryotic signal peptidase. These include specifically the length of the hydrophobic core, the location of .beta.-turns relative to the COOH terminus of this core domain, as well as the physical-chemical properties of amino acids at potential cleavage sites.

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Accession: 007316803

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