Transport of proteins into chloroplasts. The thylakoidal processing peptidase is a signal-type peptidase with stringent substrate requirements at the -3 and -1 positions.
The transport of proteins across the thylakoid membrane in higher plant chloroplasts is usually mediated by an amino-terminal peptide extension which is subsequently removed by a specific thylakoidal processing peptidase. We have previously shown that the reaction specificity of this enzyme is very similar to those of signal peptidases located in the endoplasmic reticulum and bacterial plasma membrane. In the present report, the reaction mechanism of the thylakoidal peptidase has been investigated by substituting a variety of amino acids for the alanine residues at the -3 and -1 positions of a thylakoid lumen protein precursor. Small neutral side chains are known to be essential at these positions for cleavage by signal peptidases, and we find that these residues likewise play a critical role in defining the thylakoidal processing peptidase cleavage site. However, the requirements of the thylakoidal enzyme at these sites are significantly more restrictive than those of the bacterial or endoplasmic reticulum peptidases. Whereas leucine at the -3 position in the substrate is tolerated by the latter two enzymes, cleavage by the thylakoidal peptidase is almost completely inhibited. At the -1 position the presence of alanine appears to be critical; substitution of this residue by glycine, serine, threonine, leucine, lysine, or glutamate leads to either substantial or complete inhibition of cleavage at this site. Substitutions at either -3 or -1 which blocked cleavage at the correct site led to cleavage taking place at an alternative site, probably after the -21 residue.
Shackleton
J B
JB
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
Robinson
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002736:Chloroplasts / Q000378:metabolism*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity
D011506:Proteins; D010450:Endopeptidases; C069654:thylakoid processing peptidase