Proteomic assessment shows that many endoplasmic reticulum (ER)-resident proteins are targeted by N(epsilon)-lysine acetylation in the lumen of the organelle and predicts broad biological impact

Abstract

In addition to the nucleus, cytosol, and mitochondrial lumen, N(ε)-lysine acetylation also occurs in the lumen of the endoplasmic reticulum (ER). However, the impact of such an event on ER functions is still unknown. Here, we analyzed the "ER acetyl-lysine proteome" by nano-LC-MS/MS and discovered that a large number of ER-resident and -transiting proteins undergo N(ε)-lysine acetylation in the lumen of the organelle. The list of ER-resident proteins includes chaperones and enzymes involved with post-translational modification and folding. Grouping of all acetylated proteins into major functional categories suggests that the ER-based acetylation machinery regulates very diverse biological events. As such, it is predicted to play a fundamental role in human physiology as well as human pathology.

FIGURE 1.

Overview of the ER acetyl-lysine proteome analysis. A and B, topological classification of acetylated resident (A) and transiting (B) proteins. The complete list of proteins and peptides is in supplemental Tables S1 and S2. C, functional classification of all acetylated proteins (resident and transiting). Proteins were assigned based on the Protein Analysis THrough Evolutionary Relationships (PANTHER) classification system. D, interaction network from STRING analysis of acetylated resident proteins. STRING analysis was performed using “experiments, database, and text mining evidences” as prediction methods (using STRING 9.0). Description of the current STRING database as well as analysis procedures can be found in Ref. . Thickness of the connecting lines indicates the confidence score of the association (thicker lines indicate stronger confidence). Resident proteins were grouped based on functions as follows: gray, chaperones; red, proteins involved with post-translational modification; black, proteins involved with quality control and ER-associated protein degradation; white, other.

FIGURE 2.

Validation of the acetylation status of ER-resident proteins. A, the indicated ER-resident proteins were immunoprecipitated (I. P.) prior to Western blot (W. b.) analysis with the indicated antibodies. Results are in duplicate. Ac. Lys., acetylated lysine. B and C, transgenic Myc-tagged BiP (B) or calreticulin (C) were purified with an immobilized anti-Myc column and then analyzed by LC-MS/MS to detect acetylated lysine residues. The domain organization of BiP (B; lower panel) and calreticulin (C; lower panel) with modified lysine residues is also shown. The domain organization of BiP is based on Refs. –, whereas the domain organization of calreticulin is based on Refs. and .

FIGURE 3.

Quantitative assessment of the acetylation status of BiP and calreticulin. Transgenic (Myc-tagged) BiP and calreticulin (Calr) were purified with an immobilized anti-Myc column. The affinity-purified protein (Input) was immunoprecipitated with an anti-acetylated lysine antibody (Ac-K IP) prior to Western blot assessment with the indicated antibodies. Quantitative analysis of the acetylation status of both BiP (A) and calreticulin (B) is shown. Representative Western blots are shown in the left panels, whereas the average (n = 4) ± S.D. is shown in the right panels. O. D., optical density.