A subset of phagosomes in human macrophages and dendritic cells that is marked by a coat of autophagy-related proteins maintains phagocytosed antigens for prolonged presentation on MHC class II molecules.

Immature and nonnative proteins are retained in the endoplasmic reticulum (ER) by the quality control machinery. Folding-incompetent glycoproteins are eventually targeted for ER-associated protein degradation (ERAD). EDEM1 (ER degradation-enhancing ␣-mannosidaselike protein 1), a putative mannose-binding protein, targets misfolded glycoproteins for ERAD. We report that endogenous EDEM1 exists mainly as a soluble glycoprotein. By high-resolution immunolabeling and serial section analysis, we find that endogenous EDEM1 is sequestered in buds that form along cisternae of the rough ER at regions outside of the transitional ER. They give rise to Ϸ150-nm vesicles scattered throughout the cytoplasm that are lacking a recognizable COPII coat. About 87% of the immunogold labeling was over the vesicles and Ϸ11% over the ER lumen. Some of the EDEM1 vesicles also contain Derlin-2 and the misfolded Hong Kong variant of ␣-1-antitrypsin, a substrate for EDEM1 and ERAD. Our results demonstrate the existence of a vesicle budding transport pathway out of the rough ER that does not involve the canonical transitional ER exit sites and therefore represents a previously unrecognized passageway to remove potentially harmful misfolded luminal glycoproteins from the ER.electron microscopy ͉ protein misfolding ͉ protein quality control T he folding state of newly synthesized glycoproteins in the endoplasmic reticulum (ER) is monitored by a quality control machinery (1). Increased formation of misfolded proteins disturbs ER homeostasis resulting in protein degradation, as well as cell damage and death. This is the cause of many human diseases including cystic fibrosis, ␣-1-antitrypsin deficiency, renal diabetes insipidus, and congenital goiter (2, 3).Orderly occurring processes can be distinguished during the life and death of a folding-incompetent glycoprotein. The first involves recognition by the quality control machinery (4-6). The lectin chaperones calnexin/calreticulin retain nonnative conformers with monoglucosylated glycans (5, 7). Mannose-trimmed glycans generated by ER-mannosidases appear to represent a quality control tag for routing misfolded glycoproteins to the ER-associated degradation (ERAD) (8-12). The current view is that misfolded, mannosetrimmed glycoproteins are then retrotranslocated to the cytoplasm, where they are ubiquitinated and deglycosylated before proteasomal degradation (13).EDEM1 (ER degradation-enhancing ␣-mannosidase-like protein 1) and its yeast ortholog Htm1p/Mnl1p are putative mannosebinding proteins (14-18) that are transcriptionally induced by ER stress (14,17,18). They are believed to target misfolded glycoproteins for proteasomal degradation by removing them from the calnexin/calreticulin cycle (14-17). The overexpression of EDEM1 results in the accelerated proteasomal degradation of ERAD substrates such as the Hong Kong variant of ␣-1-antitrypsin (HK A1AT) and a luminal variant of beta-secretase, whereas the deletion or knockdown of EDEM1/Htm1p reduces the degradation rates of the ERAD substrates ...

Quality control of protein folding represents a fundamental cellular activity. Early steps of protein N-glycosylation involving the removal of three glucose and some specific mannose residues in the endoplasmic reticulum have been recognized as being of importance for protein quality control. Specific oligosaccharide structures resulting from the oligosaccharide processing may represent a glycocode promoting productive protein folding, whereas others may represent glyco-codes for routing not correctly folded proteins for dislocation from the endoplasmic reticulum to the cytosol and subsequent degradation. Although quality control of protein folding is essential for the proper functioning of cells, it is also the basis for protein folding disorders since the recognition and elimination of non-native conformers can result either in loss-of-function or pathological-gain-of-function. The machinery for protein folding control represents a prime example of an intricate interactome present in a single organelle, the endoplasmic reticulum. Here, current views of mechanisms for the recognition and retention leading to productive protein folding or the eventual elimination of misfolded glycoproteins in yeast and mammalian cells are reviewed.

Trimming of N-linked oligosaccharides by endoplasmic reticulum (ER) glucosidase II is implicated in quality control of protein folding. An alternate glucosidase II-independent deglucosylation pathway exists, in which endo-␣-mannosidase cleaves internally the glucose-substituted mannose residue of oligosaccharides. By immunogold labeling, we detected most endomannosidase in cis/medial Golgi cisternae (83.8% of immunogold labeling) and less in the intermediate compartment (15.1%), but none in the trans-Golgi apparatus and ER, including its transitional elements. This dual localization became more pronounced under 15°C conditions indicative of two endomannosidase locations. Under experimental conditions when the intermediate compartment marker p58 was retained in peripheral sites, endomannosidase was redistributed to the Golgi apparatus. Double immunogold labeling established a mutually exclusive distribution of endomannosidase and glucosidase II, whereas calreticulin was observed in endomannosidasereactive sites (17.3% in intermediate compartment, 5.7% in Golgi apparatus) in addition to the ER (77%). Our results demonstrate that glucose trimming of N-linked oligosaccharides is not limited to the ER and that protein deglucosylation by endomannosidase in the Golgi apparatus and intermediate compartment additionally ensures that processing to mature oligosaccharides can continue. Thus, endomannosidase localization suggests that a quality control of N-glycosylation exists in the Golgi apparatus. INTRODUCTIONA common posttranslational modification on proteins, while being present in the endoplasmic reticulum (ER), is the addition of asparagine-linked oligosaccharides. Immediately after the transfer of the lipid-linked preassembled Glc 3 Man 9 GlcNAc 2 oligosaccharide to asparagine, the glucose residues are trimmed by the sequential action of the ER residents glucosidase I and II (reviewed in Moremen et al., 1994;Roth, 1995). Although it has been known for some time that the glucose residues are essential determinants for Nglycosylation (Spiro et al., 1979;Turco and Robbins, 1979;Murphy and Spiro, 1981) and that subsequent excision of these sugars is required for the formation of complex carbohydrate units, it is only recently that the monoglucosylated oligosaccharide has been implicated in quality control of ER-situated protein folding (reviewed in Ellgaard et al., 1999). Monoglucosylated oligosaccharide intermediate involved in this process can be generated either by glucosidase II trimming Hebert et al., 1995;Jakob et al., 1998b) or by reglucosylation through the action of UDP-Glc:glycoprotein glucosyltransferase (Trombetta and Parodi, 1992;Sousa and Parodi, 1995;Fernandez et al., 1996;Fanchiotti et al., 1998). Current evidence points to an ER control mechanism monitoring the folding state of proteins by the concerted action of UDP-Glc:glycoprotein glucosyltransferase, glucosidase II, and various chaperones, including calnexin and calreticulin (Zapun et al., 1988;Oliver et al., 1997;Zhang et al., 1997;Jakob et al., 1...