Oligomannosides or oligosaccharide-lipids as potential substrates for rat liver cytosolic α- d -mannosidase

157

137

One challenge facing eukaryotic cells is the posttranslational import of proteins into organelles. This problem is exacerbated when the proteins assemble into large complexes. Aminopeptidase I (API) is a resident hydrolase of the vacuole/lysosome in the yeast Saccharomyces cerevisiae. The precursor form of API assembles into a dodecamer in the cytosol and maintains this oligomeric form during the import process. Vacuolar delivery of the precursor form of API requires a vesicular mechanism termed the cytoplasm to vacuole targeting (Cvt) pathway. Many components of the Cvt pathway are also used in the degradative autophagy pathway. ␣-Mannosidase (Ams1) is another resident hydrolase that enters the vacuole independent of the secretory pathway; however, its mechanism of vacuolar delivery has not been established. We show vacuolar localization of Ams1 is blocked in mutants that are defective in the Cvt and autophagy pathways. We have found that Ams1 forms an oligomer in the cytoplasm. The oligomeric form of Ams1 is also detected in subvacuolar vesicles in strains that are blocked in vesicle breakdown, indicating that it retains its oligomeric form during the import process. These results identify Ams1 as a second biosynthetic cargo protein of the Cvt and autophagy pathways.

Section: Ams1 Utilizes the Autophagy Pathway For Vacuolar Delivery Unmentioning

confidence: 99%

Vacuolar Localization of Oligomeric α-Mannosidase Requires the Cytoplasm to Vacuole Targeting and Autophagy Pathway Components in Saccharomyces cerevisiae

Hutchins

Klionsky

2001

157

137

“…A third route for oligosaccharide production (III) was hypothesized to be the result of the action of an endo-␤-N-acetylglucosaminidase (ENGase) acting upon N-linked glycoproteins to give rise to OS-Gn 1 (55). More recently, a fourth reaction (IV) involving formation of OS-Gn 2 from glycoproteins by the action of a PNGase, which cleaves the amide bond between the glycosylated Asn residue and the innermost GlcNAc residue, has been proposed (56,57). Indeed, the occurrence of both ENGase and PNGase in an ER-enriched fraction has been reported (58 -60).…”

Section: Minireview: Two-way Traffic Across the Er Membrane 10084mentioning

confidence: 99%

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

Suzuki

Lennarz

1998

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...

“…These enzymes are activated by cobalt, are relatively resistant to swainsonine, and can convert Man 5 GlcNAc to Man 3 GlcNAc. However, they act efficiently only on glycans with a single GlcNAc residue on their reducing end and appear to function in N-glycan catabolism rather than processing (33)(34)(35)(36).…”

Section: Discussionmentioning

confidence: 99%

Insect Cells Encode a Class II α-Mannosidase with Unique Properties

Kawar

Karaveg

Moremen

et al. 2001

Previously, we cloned and characterized an insect (Sf9) cell cDNA encoding a class II ␣-mannosidase with amino acid sequence and biochemical similarities to mammalian Golgi ␣-mannosidase II. Since then, it has been demonstrated that other mammalian class II ␣-mannosidases can participate in N-glycan processing. Thus, the present study was performed to evaluate the catalytic properties of the Sf9 class II ␣-mannosidase and to more clearly determine its relationship to mammalian Golgi ␣-mannosidase II. The results showed that the Sf9 enzyme is cobalt-dependent and can hydrolyze Man 5 GlcNAc 2 to Man 3 GlcNAc 2 , but it cannot hydrolyze GlcNAcMan 5 GlcNAc 2 . These data establish that the Sf9 enzyme is distinct from Golgi ␣-mannosidase II. This enzyme is not a lysosomal ␣-mannosidase because it is not active at acidic pH and it is localized in the Golgi apparatus. In fact, its sensitivity to swainsonine distinguishes the Sf9 enzyme from all other known mammalian class II ␣-mannosidases that can hydrolyze Man 5 GlcNAc 2 . Based on these properties, we designated this enzyme Sf9 ␣-mannosidase III and concluded that it probably provides an alternate N-glycan processing pathway in Sf9 cells.