The Arabidopsis Chloroplast stromal N-terminome; complexities of N-terminal protein maturation and stability

Abstract

ORCID IDs: 0000-0003-4756-9125 (E.R.); 0000-0001-5402-7725 (J.K.); 0000-0002-6395-2610 (N.H.B.); 0000-0001-9536-0487 (K.J.v.W.).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-a-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.

Protein Degradation Rate in Arabidopsis thaliana Leaf Growth and Development

“…The FtsH protein set highlighted in Figure 6 are the same proteins that are highlighted in the protein domain set FtsH (IPR016135), which was one of the fastest degrading domain groups in our analysis (Table 2). These proteases have direct roles in the turnover of the photosynthetic apparatus and in plastid thylakoid formation (Zaltsman et al, 2005;Kato et al, 2012;Rowland et al, 2015). It has been reported that mRNA for FtsH1, FtsH2, FtsH5, and FtsH8 generally increases in young developing leaves (Zaltsman et al, 2005).…”

Chloroplast Proteases: Updates on Proteolysis within and across Suborganellar Compartments

“…Upon arrival in the stroma, SPP binds with a broad spectrum of specificity to the transit peptides and removes them by a single endoproteolytic reaction, with subsequent release of the mature proteins but not the transit peptides Lamppa, 1999, 2002). Recent proteome analysis showed that a substantial number of stromal proteins have multiple distinct N termini, implying the presence of multistep N-terminal processing mechanisms or simply reflecting SPP's imprecise site specificities (Rowland et al, 2015). The cleaved transit peptides are subjected to additional trimming into smaller fragments by SPP and are degraded further by PrePs and OOPs in the stroma (Teixeira and Glaser, 2013; Fig.…”

Posttranslational Control of ALA Synthesis Includes GluTR Degradation by Clp Protease and Stabilization by GluTR-Binding Protein

“…Early observations in yeast led to the formulation of the N-end rule, which states that certain amino acids, when exposed at the N terminus of a protein, act as triggers for degradation (Bachmair et al, 1986). An N-end rule for chloroplasts/plastids in plants is not known but certainly is a possibility (Apel et al, 2010;Rowland et al, 2015).…”