Selective recycling of the mannose 6-phosphate/IGF-II receptor to the trans Golgi network in vitro

Cell ◽  
1988 ◽  
Vol 55 (2) ◽  
pp. 309-320 ◽  
Author(s):  
Yukiko Goda ◽  
Suzanne R. Pfeffer
Keyword(s):  
Trans Golgi Network ◽  
Mannose 6 Phosphate ◽  
Golgi Network

scholarly journals A Novel Rab9 Effector Required for Endosome-to-TGN Transport

1997 ◽  
Vol 138 (2) ◽  
pp. 283-290 ◽  
Author(s):  
Elva Díaz ◽  
Frauke Schimmöller ◽  
Suzanne R. Pfeffer
Keyword(s):  
Active Form ◽  
Sucrose Density Gradient ◽  
Vesicle Docking ◽  
Trans Golgi Network ◽  
Yeast Two Hybrid System ◽  
Transport Assay ◽  
Transport Vesicle ◽  
Mannose 6 Phosphate ◽  
Golgi Network

Rab9 GTPase is required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network in living cells, and in an in vitro system that reconstitutes this process. We have used the yeast two-hybrid system to identify proteins that interact preferentially with the active form of Rab9. We report here the discovery of a 40-kD protein (p40) that binds Rab9–GTP with roughly fourfold preference to Rab9–GDP. p40 does not interact with Rab7 or K-Ras; it also fails to bind Rab9 when it is bound to GDI. The protein is found in cytosol, yet a significant fraction (∼30%) is associated with cellular membranes. Upon sucrose density gradient flotation, membrane- associated p40 cofractionates with endosomes containing mannose 6-phosphate receptors and the Rab9 GTPase. p40 is a very potent transport factor in that the pure, recombinant protein can stimulate, significantly, an in vitro transport assay that measures transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network. The functional importance of p40 is confirmed by the finding that anti-p40 antibodies inhibit in vitro transport. Finally, p40 shows synergy with Rab9 in terms of its ability to stimulate mannose 6-phosphate receptor transport. These data are consistent with a model in which p40 and Rab9 act together to drive the process of transport vesicle docking.

scholarly journals Identification of a novel, N-ethylmaleimide-sensitive cytosolic factor required for vesicular transport from endosomes to the trans-Golgi network in vitro.

1991 ◽  
Vol 112 (5) ◽  
pp. 823-831 ◽  
Author(s):  
Y Goda ◽  
S R Pfeffer
Keyword(s):  
Early Stage ◽  
Gradient Centrifugation ◽  
Vesicular Transport ◽  
Free System ◽  
Transport Reaction ◽  
Trans Golgi Network ◽  
Transport Vesicles ◽  
Golgi Network ◽  
Gamma S

We have recently described a cell-free system that reconstitutes the vesicular transport of 300-kD mannose 6-phosphate receptors from late endosomes to the trans-Golgi network (TGN). We report here that the endosome----TGN transport reaction was significantly inhibited by low concentrations of the alkylating agent, N-ethylmaleimide (NEM). Addition of fresh cytosol to NEM-inactivated reaction mixtures restored transport to at least 80% of control levels. Restorative activity was only present in cytosol fractions, and was sensitive to trypsin treatment or incubation at 100 degrees C. A variety of criteria demonstrated that the restorative activity was distinct from NSF, an NEM-sensitive protein that facilitates the transport of proteins from the ER to the Golgi complex and between Golgi cisternae. Cytosol fractions immunodepleted of greater than or equal to 90% of NSF protein, or heated to 37 degrees C to inactivate greater than or equal to 93% of NSF activity, were fully able to restore transport to NEM-treated reaction mixtures. The majority of restorative activity sedimented as a uniform species of 50-100 kD upon glycerol gradient centrifugation. We have termed this activity ETF-1, for endosome----TGN transport factor-1. Kinetic experiments showed that ETF-1 acts at a very early stage in vesicular transport, which may reflect a role for this factor in the formation of nascent transport vesicles. GTP hydrolysis appears to be required throughout the transport reaction. The ability of GTP gamma S to inhibit endosome----TGN transport required the presence of donor, endosome membranes, and cytosol, which may reflect a role for guanine nucleotides in vesicle budding. Finally, ETF-1 appears to act before a step that is blocked by GTP gamma S, during the process by which proteins are transported from endosomes to the TGN in vitro.

scholarly journals Lysosomal Hydrolase Mannose 6-Phosphate Uncovering Enzyme Resides in the trans-Golgi Network

2001 ◽  
Vol 12 (6) ◽  
pp. 1623-1631 ◽  
Author(s):  
Jack Rohrer ◽  
Rosalind Kornfeld
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Lysosomal Hydrolases ◽  
Intracellular Location ◽  
Trans Golgi Network ◽  
Cytosolic Domain ◽  
Cytosolic Domains ◽  
Mannose 6 Phosphate ◽  
Green Fluorescent ◽  
Golgi Network

A crucial step in lysosomal biogenesis is catalyzed by “uncovering” enzyme (UCE), which removes a coveringN-acetylglucosamine from the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal hydrolases. This study shows that UCE resides in the trans-Golgi network (TGN) and cycles between the TGN and plasma membrane. The cytosolic domain of UCE contains two potential endocytosis motifs: 488YHPL and C-terminal 511NPFKD. YHPL is shown to be the more potent of the two in retrieval of UCE from the plasma membrane. A green-fluorescent protein-UCE transmembrane-cytosolic domain fusion protein colocalizes with TGN 46, as does endogenous UCE in HeLa cells, showing that the transmembrane and cytosolic domains determine intracellular location. These data imply that the Man-6-P recognition marker is formed in the TGN, the compartment where Man-6-P receptors bind cargo and are packaged into clathrin-coated vesicles.

Syntaxin 11 is associated with SNAP-23 on late endosomes and the trans-Golgi network

1999 ◽  
Vol 112 (6) ◽  
pp. 845-854 ◽  
Author(s):  
A.C. Valdez ◽  
J.P. Cabaniols ◽  
M.J. Brown ◽  
P.A. Roche
Keyword(s):  
Mammalian Cells ◽  
Protein Transport ◽  
Transmembrane Domain ◽  
Family Members ◽  
Snare Proteins ◽  
Trans Golgi Network ◽  
Late Endosomes ◽  
Syntaxin 11 ◽  
Golgi Network

SNARE proteins are known to play a role in regulating intracellular protein transport between donor and target membranes. This docking and fusion process involves the interaction of specific vesicle-SNAREs (e.g. VAMP) with specific cognate target-SNAREs (e.g. syntaxin and SNAP-23). Using human SNAP-23 as the bait in a yeast two-hybrid screen of a human B-lymphocyte cDNA library, we have identified the 287-amino-acid SNARE protein syntaxin 11. Like other syntaxin family members, syntaxin 11 binds to the SNARE proteins VAMP and SNAP-23 in vitro and also exists in a complex with SNAP-23 in transfected HeLa cells and in native human B lymphocytes. Unlike other syntaxin family members, no obvious transmembrane domain is present in syntaxin 11. Nevertheless, syntaxin 11 is predominantly membrane-associated and colocalizes with the mannose 6-phosphate receptor on late endosomes and the trans-Golgi network. These data suggest that syntaxin 11 is a SNARE that acts to regulate protein transport between late endosomes and the trans-Golgi network in mammalian cells.

Antibodies to clathrin inhibit endocytosis but not recycling to the trans Golgi network in vitro

Science ◽  
1990 ◽  
Vol 248 (4962) ◽  
pp. 1539-1541 ◽  
Author(s):  
R. Draper ◽  
Y Goda ◽  
F. Brodsky ◽  
Pfeffer
Keyword(s):  
Trans Golgi Network ◽  
Golgi Network

scholarly journals Transport of mannose-6-phosphate receptors from the trans-Golgi network to endosomes requires Rab31

2009 ◽  
Vol 315 (13) ◽  
pp. 2215-2230 ◽  
Author(s):  
A.G. Rodriguez-Gabin ◽  
X. Yin ◽  
Q. Si ◽  
J.N. Larocca
Keyword(s):  
Trans Golgi Network ◽  
Mannose 6 Phosphate ◽  
Golgi Network

scholarly journals Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus.

1988 ◽  
Vol 106 (3) ◽  
pp. 617-628 ◽  
Author(s):  
J R Duncan ◽  
S Kornfeld
Keyword(s):  
Sialic Acid ◽  
Golgi Apparatus ◽  
Chinese Hamster ◽  
Murine Lymphoma ◽  
Trans Golgi Network ◽  
Intracellular Movement ◽  
Golgi Region ◽  
Golgi Compartment ◽  
Mannose 6 Phosphate ◽  
Golgi Network

We have used Chinese hamster ovary (CHO) cells and a murine lymphoma cell line to study the recycling of the 215-kD and the 46-kD mannose 6-phosphate receptors to various regions of the Golgi to determine the site where the receptors first encounter newly synthesized lysosomal enzymes. For assessing return to the trans-most Golgi compartments containing sialyltransferase (trans-cisternae and trans-Golgi network), the oligosaccharides of receptor molecules on the cell surface were labeled with [3H]galactose at 4 degrees C. Upon warming to 37 degrees C, the [3H]galactose residues on both receptors were substituted with sialic acid with a t1/2 approximately 3 hrs. Other glycoproteins acquired sialic acid at least 8-10 times slower. Return of the receptors to the trans-Golgi cisternae containing galactosyltransferase could not be detected. Return to the cis/middle Golgi cisternae containing alpha-mannosidase I was measured by adding deoxymannojirimycin, a mannosidase I inhibitor, during the initial posttranslational passage of [3H]mannose-labeled glycoproteins through the Golgi, thereby preserving oligosaccharides which would be substrates for alpha-mannosidase I. After removal of the inhibitor, return to the early Golgi with subsequent passage through the Golgi complex was measured by determining the conversion of the oligosaccharides from high mannose to complex-type units. This conversion was very slow for the receptors and other glycoproteins (t1/2 approximately 20 h). Exposure of the receptors and other glycoproteins to the dMM-sensitive alpha-mannosidase without movement through the Golgi apparatus was determined by measuring the loss of mannose residues from these proteins. This loss was also slow. These results indicate that both Man-6-P receptors routinely return to the Golgi compartment which contains sialyltransferase and recycle through other regions of the Golgi region less frequently. We infer that the trans-Golgi network is the major site for lysosomal enzyme sorting in CHO and murine lymphoma cells.

scholarly journals Phosphorylation of the cation-independent mannose 6-phosphate receptor is closely associated with its exit from the trans-Golgi network.

1993 ◽  
Vol 120 (1) ◽  
pp. 67-75 ◽  
Author(s):  
S Méresse ◽  
B Hoflack
Keyword(s):  
Cell Surface ◽  
Specific Inhibitor ◽  
Cytoplasmic Domain ◽  
Single Step ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
Mannose 6 Phosphate ◽  
Golgi Network

We have previously shown that two serine residues present in two conserved regions of the bovine cation-independent mannose 6-phosphate receptor (CI-MPR) cytoplasmic domain are phosphorylated in vivo (residues 2421 and 2492 of the full length bovine CI-MPR precursor). In this study, we have used CHO cells to investigate the phosphorylation state of these two serines along the different steps of the CI-MPR exocytic and endocytic recycling pathways. Transport and phosphorylation of the CI-MPR in the biosynthetic pathway were examined using deoxymannojirimycin (dMM), a specific inhibitor of the cis-Golgi processing enzyme alpha-mannosidase I which leads to the accumulation of N-linked high mannose oligosaccharides on glycoproteins. Upon removal of dMM, normal processing to complex-type oligosaccharides (galactosylation and then sialylation) occurs on the newly synthesized glycoproteins, including the CI-MPR which could then be purified and analyzed on lectin affinity columns. Phosphorylation of the newly synthesized CI-MPR was concomitant with the sialylation of its oligosaccharides and appeared as a major albeit transient modification. Phosphorylation of the cell surface CI-MPR was examined during its endocytosis as well as its return to the Golgi using antibody tagging and exogalactosylation. The cell surface CI-MPR was not phosphorylated when it entered clathrin-coated pits or when it moved to the early and late endosomes. In contrast, the surface CI-MPR was phosphorylated when it had been resialylated upon its return to the trans-Golgi network. Subcellular fractionation experiments showed that the phosphorylated CI-MPR and the corresponding kinase were found in clathrin-coated vesicles. Collectively, these results indicate that phosphorylation of the two serines in the CI-MPR cytoplasmic domain is associated with a single step of transport of its recycling pathways and occurs when this receptor is in the trans-Golgi network and/or has left this compartment via clathrin-coated vesicles.

scholarly journals Recovery of function in Chinese hamster ovary cell mutants with temperature-sensitive defects in vacuolar acidification.

1990 ◽  
Vol 110 (4) ◽  
pp. 1023-1032 ◽  
Author(s):  
C F Roff ◽  
C W Hall ◽  
A R Robbins
Keyword(s):  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Lysosomal Enzymes ◽  
Recovery Of Function ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Trans Golgi Network ◽  
Mannose 6 Phosphate ◽  
Golgi Network

After 4 h at 41 degrees C, B3853 and M311, temperature-sensitive Chinese hamster ovary cell End1 and End2 mutants, respectively, are pleiotropically defective in endocytosis and trans-Golgi network-associated activities (Roff, C. F., R. Fuchs, I. Mellman, and A. R. Robbins. 1986. J. Cell Biol. 103:2283-2297). We have measured recovery of function after return to the permissive temperature. Based on return of normal transferrin-mediated Fe uptake and sensitivity to diphtheria toxin both mutants had restored endosomal function at 10 h; based on delivery of endocytosed lysosomal enzymes to lysosomes and normal sensitivity to modeccin both had functional late endocytic organelles at 10-12 h; and based on retention of newly synthesized lysosomal enzymes and sialylation of secreted glycoproteins both had functional trans-Golgi network at 6 h. At 10 h, M311 had recovered almost all of its ability to endocytose lysosomal enzymes; B3853 required 30 h to recover fully its ability to endocytose lysosomal enzymes. Slow recovery of mannose 6-phosphate-dependent uptake in B3853 reflected altered trafficking of cation-independent mannose 6-phosphate receptors. Although B3853 had normal amounts of receptor at 6-8 h, it had greatly diminished amounts of receptor at the cell surface. Altered trafficking was also suggested by the finding that B3853 rapidly degraded receptor that had been present before the shift to the nonpermissive temperature.

scholarly journals High-Affinity Binding Of The AP-1 Adaptor Complex to Trans-Golgi Network Membranes Devoid Of Mannose 6-Phosphate Receptors

1999 ◽  
Vol 10 (3) ◽  
pp. 537-549 ◽  
Author(s):  
Yunxiang Zhu ◽  
Linton M. Traub ◽  
Stuart Kornfeld
Keyword(s):  
Coated Vesicle ◽  
Chemical Extraction ◽  
Normal Cells ◽  
High Affinity ◽  
Affinity Membrane ◽  
Docking Proteins ◽  
Mannose 6 Phosphate ◽  
Golgi Network

The GTP-binding protein ADP-ribosylation factor (ARF) initiates clathrin-coat assembly at the trans-Goli network (TGN) by generating high-affinity membrane-binding sites for the AP-1 adaptor complex. Both transmembrane proteins, which are sorted into the assembling coated bud, and novel docking proteins have been suggested to be partners with GTP-bound ARF in generating the AP-1-docking sites. The best characterized, and probably the major transmembrane molecules sorted into the clathrin-coated vesicles that form on the TGN, are the mannose 6-phosphate receptors (MPRs). Here, we have examined the role of the MPRs in the AP-1 recruitment process by comparing fibroblasts derived from embryos of either normal or MPR-negative animals. Despite major alterations to the lysosome compartment in the MPR-deficient cells, the steady-state distribution of AP-1 at the TGN is comparable to that of normal cells. Golgi-enriched membranes prepared from the receptor-negative cells also display an apparently normal capacity to recruit AP-1 in vitro in the presence of ARF and either GTP or GTPγS. The AP-1 adaptor is recruited specifically onto the TGN and not onto the numerous abnormal membrane elements that accumulate within the MPR-negative fibroblasts. AP-1 bound to TGN membranes from either normal or MPR-negative fibroblasts is fully resistant to chemical extraction with 1 M Tris-HCl, pH 7, indicating that the adaptor binds to both membrane types with high affinity. The only difference we do note between the Golgi prepared from the MPR-deficient cells and the normal cells is that AP-1 recruited onto the receptor-lacking membranes in the presence of ARF1·GTP is consistently more resistant to extraction with Tris. Because sensitivity to Tris extraction correlates well with nucleotide hydrolysis, this finding might suggest a possible link between MPR sorting and ARF GAP regulation. We conclude that the MPRs are not essential determinants in the initial steps of AP-1 binding to the TGN but, instead, they may play a regulatory role in clathrin-coated vesicle formation by affecting ARF·GTP hydrolysis.

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