ER chaperones use a protein folding and quality control glyco-code.

N-glycans act as quality control tags by recruiting lectin chaperones to assist protein maturation in the endoplasmic reticulum. The location and composition of N-glycans (glyco-code) are key to the chaperone-selection process. Serpins, a class of serine protease inhibitors, fold non-sequentially to achieve metastable active states. Here, the role of the glyco-code in assuring successful maturation and quality control of two human serpins, alpha-1 antitrypsin (AAT) and antithrombin III (ATIII), is described. We find that AAT, which has glycans near its N terminus, is assisted by early lectin chaperone binding. In contrast, ATIII, which has more C-terminal glycans, is initially helped by BiP and then later by lectin chaperones mediated by UGGT reglucosylation. UGGT action is increased for misfolding-prone disease variants, and these clients are preferentially glucosylated on their most C-terminal glycan. Our study illustrates how serpins utilize N-glycan presence, position, and composition to direct their proper folding, quality control, and trafficking.

TTC17 is an endoplasmic reticulum resident TPR-containing adaptor protein.

Protein folding, quality control, maturation, and trafficking are essential processes for proper cellular homeostasis. Around one-third of the human proteome is targeted to the endoplasmic reticulum (ER), the organelle that serves as entrance into the secretory pathway. Successful protein trafficking is paramount for proper cellular function and to that end there are many ER resident proteins that ensure efficient secretion. Here, biochemical and cell biological analysis was used to determine that TTC17 is a large, soluble, ER-localized protein that plays an important role in secretory trafficking. Transcriptional analysis identified the predominantly expressed protein isoform of TTC17 in various cell lines. Further, TTC17 localizes to the ER and interacts with a wide variety of chaperones and cochaperones normally associated with ER protein folding, quality control, and maturation processes. TTC17 was found to be significantly upregulated by ER stress and through the creation and use of TTC17-/- cell lines, quantitative mass spectrometry identified secretory pathway wide trafficking defects in the absence of TTC17. Notably, trafficking of insulin-like growth factor type 1 receptor, glycoprotein nonmetastatic melanoma protein B, clusterin, and UDP-glucose:glycoprotein glucosyltransferase 1 were significantly altered in H4 neuroglioma cells. This study defines a novel ER trafficking factor and provides insight into the protein-protein assisted trafficking in the early secretory pathway.

The Role of Endoplasmic Reticulum Chaperones in Protein Folding and Quality Control.

Molecular chaperones assist the folding of nascent chains in the cell. Chaperones also aid in quality control decisions as persistent chaperone binding can help to sort terminal misfolded proteins for degradation. There are two major molecular chaperone families in the endoplasmic reticulum (ER) that assist proteins in reaching their native structure and evaluating the fidelity of the maturation process. The ER Hsp70 chaperone, BiP, supports adenine nucleotide-regulated binding to non-native proteins that possess exposed hydrophobic regions. In contrast, the carbohydrate-dependent chaperone system involving the membrane protein calnexin and its soluble paralogue calreticulin recognize a specific glycoform of an exposed hydrophilic protein modification for which the composition is controlled by a series of glycosidases and transferases. Here, we compare and contrast the properties, mechanisms of action and functions of these different chaperones systems that work in parallel, as well as together, to assist a large variety of substrates that traverse the eukaryotic secretory pathway.

Quantitative glycoproteomics reveals cellular substrate selectivity of the ER protein quality control sensors UGGT1 and UGGT2.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) 1 and 2 are central hubs in the chaperone network of the endoplasmic reticulum (ER), acting as gatekeepers to the early secretory pathway, yet little is known about their cellular clients. These two quality control sensors control lectin chaperone binding and glycoprotein egress from the ER. A quantitative glycoproteomics strategy was deployed to identify cellular substrates of the UGGTs at endogenous levels in CRISPR-edited HEK293 cells. The 71 UGGT substrates identified were mainly large multidomain and heavily glycosylated proteins when compared to the general N-glycoproteome. UGGT1 was the dominant glucosyltransferase with a preference toward large plasma membrane proteins whereas UGGT2 favored the modification of smaller, soluble lysosomal proteins. This study sheds light on differential specificities and roles of UGGT1 and UGGT2 and provides insight into the cellular reliance on the carbohydrate-dependent chaperone system to facilitate proper folding and maturation of the cellular N-glycoproteome.

TPR-containing proteins control protein organization and homeostasis for the endoplasmic reticulum.

The endoplasmic reticulum (ER) is a complex, multifunctional organelle comprised of a continuous membrane and lumen that is organized into a number of functional regions. It plays various roles including protein translocation, folding, quality control, secretion, calcium signaling, and lipid biogenesis. Cellular protein homeostasis is maintained by a complicated chaperone network, and the largest functional family within this network consists of proteins containing tetratricopeptide repeats (TPRs). TPRs are well-studied structural motifs that mediate intermolecular protein-protein interactions, supporting interactions with a wide range of ligands or substrates. Seven TPR-containing proteins have thus far been shown to localize to the ER and control protein organization and homeostasis within this multifunctional organelle. Here, we discuss the roles of these proteins in controlling ER processes and organization. The crucial roles that TPR-containing proteins play in the ER are highlighted by diseases or defects associated with their mutation or disruption.