Identification and analysis of the acetylated status of poplar proteins reveals analogous N-terminal protein processing mechanisms with other eukaryotes

Abstract

Background: The N-terminal protein processing mechanism (NPM) including N-terminal Met excision (NME) and N-terminal acetylation (N(α)-acetylation) represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown.

Methodology/principal findings: To reveal the NPM in poplar, we investigated the N(α)-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO2 enrichment of acetylated peptides. We identified 58 N-terminally acetylated (N(α)-acetylated) proteins. Most proteins (47, >81%) are subjected to N(α)-acetylation following the N-terminal removal of Met, indicating that N(α)-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and N(α)-acetylation (NPM) to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs) and N-terminal acetyltransferase (Nat) enzymes in poplar. The N(α)-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins.

Conclusions/significance: This study represents the first extensive investigation of N(α)-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of N(α)-acetylation of proteins in poplar.

Figure 1. Phylogenetic analysis of MAPs.

A phylogenetic tree was generated with the neighbor-joining method using the p-distance in MEGA version 5.04 from all complete MAP protein sequences of Arabidopsis and poplar. Branch lengths are proportional to the amino acid distances along each branch. The neighbor-joining bootstrap values for clusters supported above the 50% level are indicated in the red font above the branches, whereas minimum-evolution bootstrap values above the 50% level are presented in red font below the branches. The blue diamonds are highlighted at the front of all MAPs from Populus. PtrMAP1A (XP_002320050.1); Ptr MAP1B (XP_002311439.1); Ptr MAP1C (JGI protein ID, 225648); Ptr MAP1D (XP_002306464.1); Ptr MAP1E (XP_002336218.1); Ptr MAP2A (XP_002301676.1); Ptr MAP2B (XP_002305888.1); Ath MAP1A (AAP37849.1); Ath MAP1B (AEE28993.1); Ath MAP1C (AEE77063.1); Ath MAP1D (AEE86740.1); Ath MAP2B (AEE79999.1); Ath MAP2A (AAP21284.1).

Figure 2. These identified protein acetylation reaction and sequence alignment of the acetylation sites.

A, the acetylation reaction and sequence alignment of the 11 N^α-acetylated proteins that retained their Met residue from group (i). B, the acetylation reaction and sequence alignment of the acetylated sites of the 47 N^α-acetylation proteins where the Met residue has been removed from group (ii). “-AC” represents N^α-acetylation, and in B, the blank at position 1 represents the removed of the N-terminal Met residue; sequence alignment of the acetylation sites was made by Weblogo program.