Protein N-termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts, essential organelles in plants. 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 using terminal amine isotopic labeling of substrates (TAILS) and mass spectrometry. Many nuclear-encoded plastid proteins accumulated with two or three different N-termini, assigned ‘ragged ends‘. Ala, Val, Thr (often in N-α acetylated form) and Ser 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 under-represented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of Met. Infrequent residues (e.g. Ile, Arg, Cys, Pro, Asp, Glu) were observed for several abundant proteins (e.g. HSP70/90, RBCL, Fd-GOGAT) 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 (Asp, Glu, Leu, Phe). This suggest that a N-end rule might  contribute to chloroplast stromal but not lumenal proteostasis. Consistently, chloroplast posses an elaborate stromal Clp protease system with ClpC/D chaperones, a heteromeric Clp core complex and a ClpS adaptor that interacts not only with ClpC but also with a novel and plant specific co-adaptor ClpF.  We discuss a possible N-end rule in plant chloroplasts.

Complexities of N-terminal protein maturation and stability in chloroplasts

255th National Meeting (2018)

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