The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability

Plant Physiol. 2015 Nov;169(3):1881-96. doi: 10.1104/pp.15.01214. Epub 2015 Sep 14.

Abstract

Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Oxidoreductases / genetics
  • Amino Acid Oxidoreductases / metabolism
  • Amino Acid Sequence
  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Chloroplast Proteins / genetics
  • Chloroplast Proteins / metabolism
  • Chloroplasts / metabolism
  • Metalloendopeptidases / genetics
  • Metalloendopeptidases / metabolism
  • Models, Biological
  • Molecular Sequence Data
  • Plant Proteins / genetics
  • Plant Proteins / metabolism
  • Protein Processing, Post-Translational*
  • Protein Stability
  • Proteome*
  • Ribulose-Bisphosphate Carboxylase / genetics
  • Ribulose-Bisphosphate Carboxylase / metabolism

Substances

  • Arabidopsis Proteins
  • Chloroplast Proteins
  • Plant Proteins
  • Proteome
  • Amino Acid Oxidoreductases
  • glutamate synthase (ferredoxin)
  • Metalloendopeptidases
  • stromal processing peptidase
  • Ribulose-Bisphosphate Carboxylase