Extent ofN-terminal methionine excision fromEscherichia coli proteins isgoverned bytheside-chain length ofthe penultimate aminoacid (protein maturation/methionyl-aminopeptidase/hybrid protein expression)

Inasignificant fraction oftheEscherichia coli cytosolic proteins, theN-terminal methionine residue incorpo- rated during thetranslation initiation stepisexcised. The N-terminal methionine excision iscatalyzed bymethionyl- aminopeptidase (MAP). Previous studies havesuggested that theaction ofthis enzymecould depend mainly onthenature of thesecond aminoacid residue inthepolypeptide chain. Inthis study, toachieve asystematic analysis ofthespecificity ofMAP action, eachofthe20aminoacids wasintroduced atthe penultimate position ofmethionyl-tRNA synthetase ofE.coli andtheextent ofinvivo methionine excision wasmeasured. To facilitate variant protein purification andN-terminal sequence determination, anexpression shuttle vector based onprotein fusion with(3-galactosidase wasused. Fromourresults, me- thionine excision catalyzed byMAP isshowntoobeythe following rule: thecatalytic efficiency ofMAP,andtherefore theextent ofcleavage, decreases inparallel withtheincreasing ofthemaximal side-chain length oftheaminoacidinthe penultimate position. Thismolecular modelaccounts forthe rateofN-terminal methionine excision inE.coli, asdeduced fromtheanalysis of100protein N-terminal sequences. Incorporation ofamethionine residue attheN terminus of eachnascent polypeptide makespart oftheuniversal trans- lation initiation signal, usedbyprokaryotes aswellaseu- karyotes. Inprokaryotes, themethionyl moiety carried by theinitiator tRNAisN-formylated prior toits incorporation. However, soluble proteins retaining aformylated Nterminus donotrepresent ameasurable fraction oftotal proteins in Escherichia coli (1,2). Moreover, inacytosolic extract ofE. coli, only 40%ofthepolypeptidic chains retain anN-terminal methionine. Instead, about 50%display alanine, serine, or threonine attheir N termini (3). Theseobservations areaccounted forbyearly posttrans- lational modifications ofthepolypeptides. Theformyl group andmethionine residue areremoved sequentially, thede- formylation step being moretightly coupled tothetranslation process thanthemethionine excision (4). Theoccurrence of twoseparate activities wasestablished bythepurification of theE.coli deformylase enzyme(2,5-7)and,veryrecently, bythecloning ofthemethionyl-aminopeptidase