ABSTRACT
The Nε-lysine acetylation of cargo proteins in the lumen of the endoplasmic reticulum (ER) requires a membrane transporter (SLC33A1) and 2 acetyltransferases (NAT8B and NAT8). The ER acetylation machinery regulates the homeostatic balance between quality control/efficiency of the secretory pathway and autophagy-mediated disposal of toxic protein aggregates. We recently reported that the autophagy pathway that acts downstream of the ER acetylation machinery specifically targets protein aggregates that form within the secretory pathway. Genetic and biochemical manipulation of ER acetylation in a mouse model of Alzheimer disease is able to restore normal proteostasis and rescue the disease phenotype. Here we summarize these findings and offer an overview of the ER-acetylation machinery.
KEYWORDS: acetyl-CoA, autophagy, endoplasmic reticulum, lysine acetylation, secretory pathway
Nε-lysine acetylation was initially identified on histones, cytoskeletal proteins and transcription factors. For more than 40 years, it was assumed it only occurred in the cytosol and nucleus. However, in 2007 our group reported the transient lysine acetylation of BACE1 (β-site APP-cleaving enzyme 1), an endoplasmic reticulum (ER)-transiting protein that is involved in the pathogenesis of Alzheimer disease (AD). Specifically, we discovered that BACE1 is acetylated in the ER and deacetylated in the Golgi apparatus, while trafficking along the secretory pathway. Site-directed mutagenesis showed that acetylated intermediates of BACE1 are able to move through the secretory pathway with high efficiency while nonacetylated intermediates are retained and disposed of. Mass spectrometry identified 7 lysine residues being acetylated; all of them were positioned in the N-terminal ectodomain of the protein facing the lumen of the ER. Therefore, against our expectations, the Nε-lysine acetylation of the nascent protein occurs in the lumen of the organelle. Subsequent proteomic studies from 2 laboratories (our own and that of Matthias Mann at the Max Planck Institute, Martinsried, Germany) identified several ER-transiting and -resident proteins as being acetylated in the lumen of the ER. Therefore, intralumenal Nε-lysine acetylation is a previously unknown intrinsic feature of ER biology. Finally, while studying PROM1/CD133, a marker of cancer stem cells, the laboratory of Jason Moffat (University of Toronto, Toronto, Canada) confirmed the early conclusions obtained with BACE1. Even in this case, the transient acetylation of CD133 in the ER lumen is able to regulate efficiency of trafficking of the nascent protein to the cell surface.
The fact that Nε-lysine acetylation could occur in the lumen of the ER posed an immediate biochemical challenge: in order for the reaction to occur, both the enzyme (acetyltransferase) and the donor of the acetyl group (acetyl-CoA) have to be available in the lumen of the organelle. Subsequent efforts from our group resulted in the identification of 2 ER-based acetyltransferases, NAT8B/ATase1 and NAT8/ATase2. Follow-up studies showed that SLC33A1/AT-1 is the ER membrane acetyl-CoA transporter.
One of the functions of the ER is to ensure that nascent membrane and secreted polypeptides fold correctly. Although the essential information for folding is present in the primary amino acid sequence, co-translational events (such as N-glycosylation and disulfide bond formation) help to preserve the fidelity of the process. Incorrectly folded polypeptides, which fail quality control, must be “sorted” and disposed of. For this purpose, transient post-translational modifications have been designed to “select” correctly folded and unfolded/misfolded polypeptides.
While characterizing the assembly state of NAT8B and NAT8, we discovered that they interact with the oligosaccharyl transferase complex during the translocation of nascent polypeptides through the ER membrane, and acetylate correctly folded polypeptides. Targeted mutagenesis revealed that only correctly folded nascent polypeptides are recognized by NAT8B/NAT8 and acetylated; the acetylated polypeptides are able to move through the secretory pathway while non-acetylated polypeptides are not. Therefore, the ER-acetylation machinery appears to function as a “positive” selection system that recognizes correctly folded polypeptides.
Another important function of the ER is to dispose of unfolded/misfolded polypeptides. Monomeric proteins are preferentially degraded by the proteasome. In contrast, large protein aggregates are mostly dealt with by activating autophagy. While trying to characterize the biology of SLC33A1, we discovered that the influx of acetyl-CoA into the ER lumen regulates the induction of autophagy. Subsequent studies in a mouse model of SLC33A1 haploinsufficiency (SLC33A1S113R/+/AT-1S113R/+ mice) confirmed these conclusions. At the mechanistic level, the regulation of autophagy involves intralumenal acetylation of the autophagy protein ATG9A, which appears to act as a “sensor” for the acetylation status of the ER. Importantly, the expression of SLC33A1 is controlled by the unfolded protein response.
In conclusion, the ER acetylation machinery maintains the homeostatic balance of 2 essential and intimately related functions of the ER: (i) “positive” selection of correctly folded nascent polypeptides (as part of quality control) and (ii) tight regulation of autophagy (see Fig. 1).
Figure 1.
The ER acetylation machinery controls the homeostatic balance between quality control and autophagy. SLC33A1 translocates acetyl-CoA into the ER lumen. NAT8B and NAT8 transfer the acetyl group from acetyl-CoA to nascent ER polypeptides. Specifically, they associate with the oligosaccharyl transferase (OST) complex to acetylate (Ac-K) correctly folded polypeptides. If acetylated, the nascent protein is allowed to leave the ER. Unfolded/misfolded polypeptides are not acetylated by NAT8B/NAT8; as a result, they are prevented from leaving the ER and are disposed of. The ER-based acetylation also regulates the induction of autophagy through ATG9A: acetylated ATG9A prevents the induction of autophagy while nonacetylated ATG9A stimulates it. Under normal conditions, the ER acetylation machinery maintains the homeostatic balance between quality control and the induction of autophagy. When downregulated (reduced ER acetylation), the balance switches to aberrant/excessive activation of autophagy. In contrast, when upregulated (increased ER acetylation), the balance switches to excessive efficiency of the secretory pathway.
Autophagy is an essential component of the cell-degrading machinery. It helps dispose of large toxic protein aggregates that form within the secretory pathway as well as in the cytosol. Malfunction of autophagy contributes to the progression of many chronic diseases. In addition, many chronic degenerative diseases are characterized by the aberrant accumulation of toxic protein aggregates. Compelling data indicate that both hypoactive and hyperactive autophagy can be detrimental for the organism. The same data also indicate that increased levels of autophagy, which are pathogenic in wild-type mice in the absence of toxic protein aggregates, can be beneficial in mouse models of diseases characterized by increased accumulation of toxic protein aggregates. In essence, what appears to matter for the cell is the homeostatic balance between rate-of-formation and rate-of-disposal of toxic protein aggregates rather than the absolute levels of each event. Improved autophagic functions have been associated with more efficient protein and organelle homeostasis, cytoprotection, life-span extension, and rescue of proteotoxic phenotypes.
Studies in cellular systems and mouse models of dysfunctional proteostasis indicate that the autophagy pathway that acts downstream of the ER acetylation machinery specifically targets protein aggregates that form within the secretory pathway. Consistently, genetic or biochemical inhibition of the ER acetylation machinery in the mouse rescued the AD, but not the Huntington disease (HD) or the amyotrophic lateral sclerosis (ALS), phenotype. The difference between AD, HD, and ALS appears to be in the fact that the pro-aggregating and disease-triggering proteins are synthesized in different locations: APP (for AD) inserts into the secretory pathway while HTT (for HD) and SOD (for ALS) do not. These results add an additional layer of complexity to our understanding of how the cell regulates the induction and progression of autophagy. It is obvious that the cell knows where autophagy is needed and can activate it with high specificity; however, what remains to be determined is (i) how this specificity is ensured and (ii) whether we can manipulate it for translational purposes. Further characterization of the mechanisms that regulate ER acetylation will likely yield more insights on the autophagy machinery and its relationship with diseases.
Funding
This work was supported by VA Merit Award (BX001638) and NIH (NS094154 and AG033514).
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.