scholarly journals Mammalian GGAs act together to sort mannose 6-phosphate receptors

2003 ◽  
Vol 163 (4) ◽  
pp. 755-766 ◽  
Author(s):  
Pradipta Ghosh ◽  
Janice Griffith ◽  
Hans J. Geuze ◽  
Stuart Kornfeld
Keyword(s):  
Protein Trafficking ◽  
Immunogold Electron Microscopy ◽  
Partial Redistribution ◽  
Golgi Membranes ◽  
Vitro Binding ◽  
Mannose 6 Phosphate ◽  
Golgi Network ◽  
Adp Ribosylation Factor ◽  
Chemical Cross Linking

The GGAs (Golgi-localized, γ ear–containing, ADP ribosylation factor–binding proteins) are multidomain proteins implicated in protein trafficking between the Golgi and endosomes. We examined whether the three mammalian GGAs act independently or together to mediate their functions. Using cryo-immunogold electron microscopy, the three GGAs were shown to colocalize within coated buds and vesicles at the trans-Golgi network (TGN) of HeLa cells. In vitro binding experiments revealed multidomain interactions between the GGAs, and chemical cross-linking experiments demonstrated that GGAs 1 and 2 form a complex on Golgi membranes. RNA interference of each GGA resulted in decreased levels of the other GGAs and their redistribution from the TGN to cytosol. This was associated with impaired incorporation of the cation-independent mannose 6-phosphate receptor into clathrin-coated vesicles at the TGN, partial redistribution of the receptor to endosomes, and missorting of cathepsin D. The morphology of the TGN was also altered. These findings indicate that the three mammalian GGAs cooperate to sort cargo and are required for maintenance of TGN structure.

scholarly journals The Palmitoyltransferase of the Cation-dependent Mannose 6-Phosphate Receptor Cycles between the Plasma Membrane and Endosomes

2004 ◽  
Vol 15 (6) ◽  
pp. 2617-2626 ◽  
Author(s):  
Jacqueline Stöckli ◽  
Jack Rohrer
Keyword(s):  
Plasma Membrane ◽  
Protein Trafficking ◽  
Specific Protein ◽  
Cysteine Residue ◽  
Endocytic Pathway ◽  
Cell Homogenate ◽  
Mannose 6 Phosphate ◽  
Golgi Network

The cation-dependent mannose 6-phosphate receptor (CD-MPR) mediates the transport of lysosomal enzymes from the trans-Golgi network to endosomes. Evasion of lysosomal degradation of the CD-MPR requires reversible palmitoylation of a cysteine residue in its cytoplasmic tail. Because palmitoylation is reversible and essential for correct trafficking, it presents a potential regulatory mechanism for the sorting signals within the cytoplasmic domain of the CD-MPR. Characterization of the palmitoylation performing an in vitro palmitoylation assay by using purified full-length CD-MPR revealed that palmitoylation of the CD-MPR occurs enzymatically by a membrane-bound palmitoyltransferase. In addition, analysis of the localization revealed that the palmitoyltransferase cycles between endosomes and the plasma membrane. This was identified by testing fractions from HeLa cell homogenate separated on a density gradient in the in vitro palmitoylation assay and further confirmed by in vivo labeling experiments by using different treatments to block specific protein trafficking steps within the cell. We identified a novel palmitoyltransferase activity in the endocytic pathway responsible for palmitoylation of the CD-MPR. The localization of the palmitoyltransferase not only fulfills the requirement of our hypothesis to be a regulator of the intracellular trafficking of the CD-MPR but also may affect the sorting/activity of other receptors cycling through endosomes.

scholarly journals Lowe Syndrome Protein OCRL1 Interacts with Clathrin and Regulates Protein Trafficking between Endosomes and the Trans-Golgi Network

2005 ◽  
Vol 16 (8) ◽  
pp. 3467-3479 ◽  
Author(s):  
Rawshan Choudhury ◽  
Aipo Diao ◽  
Fang Zhang ◽  
Evan Eisenberg ◽  
Agnes Saint-Pol ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Lowe Syndrome ◽  
Trans Golgi Network ◽  
Partial Redistribution ◽  
Oculocerebrorenal Syndrome ◽  
Early Endosomes ◽  
Golgi Network ◽  
Syndrome Protein ◽  
In Vitro Interaction

Oculocerebrorenal syndrome of Lowe is caused by mutation of OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase localized at the Golgi apparatus. The cellular role of OCRL1 is unknown, and consequently the mechanism by which loss of OCRL1 function leads to disease is ill defined. Here, we show that OCRL1 is associated with clathrin-coated transport intermediates operating between the trans-Golgi network (TGN) and endosomes. OCRL1 interacts directly with clathrin heavy chain and promotes clathrin assembly in vitro. Interaction with clathrin is not, however, required for membrane association of OCRL1. Overexpression of OCRL1 results in redistribution of clathrin and the cation-independent mannose 6-phosphate receptor (CI-MPR) to enlarged endosomal structures that are defective in retrograde trafficking to the TGN. Depletion of cellular OCRL1 also causes partial redistribution of a CI-MPR reporter to early endosomes. These findings suggest a role for OCRL1 in clathrin-mediated trafficking of proteins from endosomes to the TGN and that defects in this pathway might contribute to the Lowe syndrome phenotype.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

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.

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 Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion.

1992 ◽  
Vol 119 (5) ◽  
pp. 1137-1150 ◽  
Author(s):  
Y A Ioannou ◽  
D F Bishop ◽  
R J Desnick
Keyword(s):  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Significant Proportion ◽  
Enzyme Concentration ◽  
Acid Sphingomyelinase ◽  
Mannose 6 Phosphate ◽  
Alpha Galactosidase ◽  
Golgi Network ◽  
Total Enzyme

Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity-purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal alpha-N-acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted alpha-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of this compartment causes the formation of soluble and particulate enzyme aggregates. A significant proportion of these enzyme aggregates are unable to bind the M6PR and are selectively secreted via the constitutive secretory pathway, while endogenous lysosomal enzymes bind the M6PRs and are transported to lysosomes.

In vitro binding of HFE to the cation-independent mannose-6 phosphate receptor

2009 ◽  
Vol 43 (2) ◽  
pp. 180-193 ◽  
Author(s):  
Lisa M. Schimanski ◽  
Hal Drakesmith ◽  
Emma Sweetland ◽  
Judy Bastin ◽  
Dellel Rezgui ◽  
...  
Keyword(s):  
In Vitro Binding ◽  
Vitro Binding ◽  
Mannose 6 Phosphate

scholarly journals A Functional Role for the GCC185 Golgin in Mannose 6-Phosphate Receptor Recycling

2006 ◽  
Vol 17 (10) ◽  
pp. 4353-4363 ◽  
Author(s):  
Jonathan V. Reddy ◽  
Alondra Schweizer Burguete ◽  
Khambhampaty Sridevi ◽  
Ian G. Ganley ◽  
Ryan M. Nottingham ◽  
...  
Keyword(s):  
Living Cells ◽  
Receptor Recycling ◽  
Transport Vesicles ◽  
Function Loss ◽  
Transport Vesicle ◽  
Enhanced Secretion ◽  
Mannose 6 Phosphate ◽  
Golgi Network ◽  
Specific Pathway

Mannose 6-phosphate receptors (MPRs) deliver newly synthesized lysosomal enzymes to endosomes and then recycle to the Golgi. MPR recycling requires Rab9 GTPase; Rab9 recruits the cytosolic adaptor TIP47 and enhances its ability to bind to MPR cytoplasmic domains during transport vesicle formation. Rab9-bearing vesicles then fuse with the trans-Golgi network (TGN) in living cells, but nothing is known about how these vesicles identify and dock with their target. We show here that GCC185, a member of the Golgin family of putative tethering proteins, is a Rab9 effector that is required for MPR recycling from endosomes to the TGN in living cells, and in vitro. GCC185 does not rely on Rab9 for its TGN localization; depletion of GCC185 slightly alters the Golgi ribbon but does not interfere with Golgi function. Loss of GCC185 triggers enhanced degradation of mannose 6-phosphate receptors and enhanced secretion of hexosaminidase. These data assign a specific pathway to an interesting, TGN-localized protein and suggest that GCC185 may participate in the docking of late endosome-derived, Rab9-bearing transport vesicles at the TGN.

scholarly journals Binding of exocytic vesicles from MDCK cells to microtubules in vitro

1990 ◽  
Vol 95 (4) ◽  
pp. 545-553
Author(s):  
P. Van der Sluijs ◽  
M.K. Bennett ◽  
C. Antony ◽  
K. Simons ◽  
T.E. Kreis
Keyword(s):  
Mdck Cells ◽  
Heat Inactivation ◽  
Microtubule Associated Proteins ◽  
Negative Stain ◽  
Vitro Binding ◽  
Viral Glycoproteins ◽  
Associated Proteins ◽  
Negative Stain Electron Microscopy ◽  
Golgi Network

Microtubules have been implicated in the transport of vesicles carrying newly synthesized proteins from the trans-Golgi network (TGN) to the cell surface. We have established a quantitative in vitro binding assay to investigate the putative interaction between these exocytic carrier vesicles and the microtubules at the molecular level. TGN-derived exocytic carrier vesicles, labeled with C6NBD-ceramide metabolites or viral glycoproteins, were obtained from polarized filter-grown MDCK II cells by perforation of the apical membrane with a nitrocellulose filter. These exocytic vesicles were incubated with taxol-polymerized tubulin and cytosol, layered on top of a 30% sucrose cushion and subjected to centrifugation. Quantitation of vesicles co-sedimenting with microtubules was done by measuring NBD-fluorescence of viral glycoproteins in the pellet and supernatant fractions. About 25% of the label sedimented through the cushion in the presence of microtubules and cytosol. Both apically and basolaterally targetted carrier vesicles containing influenza virus HA2 or vesicular stomatitis virus G protein, respectively, associated with the microtubules. Only 2–5% NBD-fluorescence was obtained in the pellet when no cytosol or microtubules were added to the vesicles. Negative-stain electron microscopy of resuspended pellets showed distinct microtubule-vesicle complexes. Heat inactivation or treatment of cytosol with N-ethylmaleimide (NEM), or trypsinization of vesicles inhibited the binding of vesicles to microtubules. Furthermore, coating of microtubules with brain microtubule-associated proteins abolished binding. These data suggest that NEM-sensitive cytosolic proteins are required for microtubule-vesicle association, and that the vesicles are bound via trypsin-sensitive receptor proteins on their surface.

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