Complex, Two-way Traffic of Molecules Across the Membrane of the Endoplasmic Reticulum

Abstract

It has been known for many years that the endoplasmic reticulum (ER) 1 is the site of assembly of polypeptide chains destined either for secretion or routing into various subcellular compartments. The N-glycosylation of these proteins, as well as their maturation assisted by certain resident luminal proteins, also occurs in the ER. Although many features of the import processes involved in synthesis of these glycoproteins have been elucidated, what is now becoming apparent is that the ER is not only involved in translocation and import, but it also functions in novel processes that mediate the export of a diversity of molecules, including unfolded or misfolded glycoproteins, glycopeptides, and oligosaccharides into the cytosol. Thus, it is clear that two-way traffic occurs, involving not only movement of molecules from the cytosol into the lumen of the ER but also out of the lumen into the cytosol. In this review the components involved in import of proteins into the ER, which have been reviewed elsewhere (1-3), are considered only in so far as they are implicated in the retrograde process, namely export out of the ER to the cytosol. Protein Import and N-Glycosylation of Proteins in the ERProtein import from the cytosol to the lumen of the ER, the first step in the biosynthesis of luminal and/or secretory proteins, occurs by either a co-or post-translational process (1-3). In both cases, proteins are known to cross the membrane by a protein-conducting channel, the translocon, in which the Sec61p trimeric complex is believed to play a central role in mammalian cells. During the co-translocational insertion of proteins into the ER the enzyme oligosaccharyltransferase (OST) transfers an oligosaccharyl moiety, in most cases GlcNAc 2 Man 9 Glc 3 , from the dolichol intermediate to Asn residues located within the sequence -Asn-X-Ser/Thr-to form Nlinked glycans on the nascent polypeptide chain. The enzyme complex has been characterized both in mammals and yeast; four subunits have been identified in the mammalian system, and so far eight subunits have been found in Saccharomyces cerevisiae (4). The spatial relationship of this enzyme with respect to the translocon remains to be determined. Also still unknown is the process whereby the oligosaccharide lipid, Dol-PP-GlcNAc 2 Man 9 Glc 3 , that serves as donor of its oligosaccharide chain in the OST-catalyzed reaction is assembled. This assembly process is believed to involve translocation within the ER membrane, i.e. "flip-flop" of some of the intermediates leading to formation of Dol-PP-GlcNAc 2 Man 9 Glc 3 . The currently accepted model is that assembly of the lipid-linked oligosaccharide occurs at the membrane of the ER in a stepwise manner (5-8), and three molecules are implicated in translocation from the cytosol to the lumen, namely Dol-PP-GlcNAc 2 Man 5 , Dol-PMan, and Dol-P-Glc. However, so far putative "flippases" to facilitate this energetically unfavorable process have not been identified. In addition, there is no information on the topological orientation o...

Endo-β- N -acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol

“…Although the cytosolic ENGase had previously been detected and biochemically characterized (20)(21)(22)(23)(24), the gene encoding this cytosolic enzyme has not been reported. There are two types of cytosolic deglycosylating enzymes that can generate free OSs from glycoproteins (7,8). One is ENGase described in this study, and the other is PNGase (peptide: N-glycanase), which releases a free glycan from glycoprotein͞ glycopeptide by cleaving the amide bond between the proximal GlcNAc residue and the side chain of an asparagine residue (35).…”

“…In recent years, it has been found that, during the N-glycosylation of proteins in the ER, a significant number of unconjugated, free oligosaccharides (free OSs) are formed (7)(8)(9). The occurrence of free OSs has been reported in a wide variety of cells (10)(11)(12)(13)(14), and their sequential processing event of free OSs in mammalian cells has also been well characterized (15)(16)(17)(18)(19).…”

“…Endo-␤-N-acetylglucosaminidase (ENGase, EC 3.2.1.96) is one of the key enzymes in the processing event of free OSs in the cytosol (8). This cytosolic enzyme activity has been described in a wide variety of animal cells (20)(21)(22)(23)(24).…”

The protein disulphide-isomerase family: unravelling a string of folds

“…This haziness in detail has, until recently, been an unfortunate characteristic of our knowledge of the crucial oxidative\non-oxidative folding processes that occur in this cellular compartment. Recent insight into the cell's response to the accumulation of misfolded protein in the ER [1], the role played by the lectins calreticulin and calnexin [2,3], as well as the mechanism of reverse transport of malfolded proteins into the cytoplasm for degradation by the proteasome [4][5][6], is shedding invaluable light on the matter. Part of the puzzle is the role played in protein folding by protein disulphideisomerase (PDI) and members of the PDI family.…”

Developmentally Regulated Expression of a Peptide:N-Glycanase during Germination of Rice Seeds (Oryza sativa) and Its Purification and Characterization

“…Ever since we unveiled the presence of PNGase and proposed its functional significance in animal cells, an increasing number of reports have appeared to implicate a possible involvement of PNGase in a wide range of biological phenomena (18 -22). PNGase activities have also been identified in Saccharomyces cerevisiae (23), and the involvement of PNGase was proposed in the mechanism of degradation of misfolded proteins that are extruded from the endoplasmic reticulum to the cytosol (24).…”