A guanosine diphosphatase enriched in Golgi vesicles of Saccharomyces cerevisiae. Purification and characterization.

129

Abstract. Golgi a-mannosidase II (G1cNAc transferase 1-dependent a1,3[a1,6] mannosidase, EC 3.2.1.114) catalyzes the final hydrolytic step in the N-glycan maturation pathway acting as the committed step in the conversion of high mannose to complex type structures . We have isolated overlapping clones from a murine cDNA library encoding the full length a-mannosidase II open reading frame and most of the 5' and 3' untranslated region . The coding sequence predicts a type II transmembrane protein with a short cytoplasmic tail (five amino acids), a single transmembrane domain (21 amino acids), and a large COOH-terminal catalytic domain (1,124 amino acids) . This domain organization which is shared with the Golgi glycosyltransferases suggests that the common structural motifs may have a functional role in Golgi enzyme function or localization .Three sets of polyadenylated clones were isolated extending 3' beyond the open reading frame by as N AND O-GLYCAN structures are increasingly being found to contribute to biological recognition events during development, oncogenic transformation, and cell adhesion (3,10,11,37,38,40) . The enzymes involved in the maturation of cell surface and intracellular N-glycans are found in the endoplasmic reticulum and Golgi complex where they act upon newly synthesized glycoproteins to generate an array of different structures from a common oligosaccharide precursor (18). The N-glycan processing pathway consists of three stages : (a) the initial synthesis of a dolichol-linked precursor oligosaccharide and the en bloc transfer of the oligosaccharide to newly synthesized polypeptide Asn-X-Ser/Thr sequons on the lumenal face of the ER; (b) the trimming of the high mannose structures by a-glucosidases and a-mannosidases in the ER and Golgi complex; and (c) the elaboration of the branched oligosaccharide chains by Golgi glycosyltransferases . The trimming phase of the pathway is accomplished by a-glucosidases I and II as well as a collection of processing a1,2-mannosidases (29) in the ER and Golgi complex. The resulting Man5GlcNAc2 structure is then modified by the addition of © The Rockefeller University Press, 0021-9525/91/12/1521/14 $2 .00 The Journal of Cell Biology, Volume 115, Number 6, December 19911521-1534 1521 much as 2,543 bp . Northern blots suggest that these polyadenylated clones totaling 6.1 kb in length correspond to minor message species smaller than the full length message. The largest and predominant message on Northern blots (7.5 kb) presumably extends another N1.4-kb downstream beyond the longest of the isolated clones. Transient expression of the a-mannosidase II cDNA in COS cells resulted in 8-12-fold overexpression of enzyme activity, and the appearance of crossreactive material in a perinuclear membrane array consistent with a Golgi localization . A region within the catalytic domain of the a-mannosidase II open reading frame bears a strong similarity to a corresponding sequence in the rat liver endoplasmic reticulum a-mannosidase and the vacuolar a-mannosidase of Sac...

Section: Discussionmentioning

confidence: 99%

Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans.

Moremen

Robbins

1991

129

“…Relative levels of specific proteins in each fraction were quantified by densitometric scanning of immunoblots. GDPase activity (Golgi marker) was determined as described (Yanagisawa et al, 1990) using CDP to subtract nonspecific phosphatase activity. Sucrose concentrations of individual fractions were determined by measuring the refractive index with an Abbe refractometer (American Optical).…”

Section: Subcellular Fractionationmentioning

confidence: 99%

Asymmetric Requirements for a Rab Gtpase and Snare Proteins in Fusion of Copii Vesicles with Acceptor Membranes

Cao

Barlowe

2000

135

138

Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion.

“…The GDPase assay was performed as described by Yanagisawa et al (1990). Samples (5 l) were mixed with 50 l 20 mM imidazole-HCl, pH 7.4, 2 mM CaCl 2 , 0.1% Triton X-100, and 5 mM GDP or CDP as control and incubated for 20 min at 30ЊC.…”

Section: Gdpase Enzyme Assaymentioning

confidence: 99%

Reconstitution of Retrograde Transport from the Golgi to the ER In Vitro

Spang

Schekman

1998

126

135

Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone α-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369–377). Retrieval depends on the HDEL sequence; the α-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.