Dynamic palmitoylation of lymphoma proprotein convertase prolongs its half-life, but is not essential for trans-Golgi network localization

Proprotein convertases are responsible for the endoproteolytic activation of proproteins in the secretory pathway. The most recently discovered member of this family, lymphoma proprotein convertase (LPC), is a type-I transmembrane protein. Previously, we have demonstrated that its cytoplasmic tail is palmitoylated. In this study, we have identified the two most proximal cysteine residues in the cytoplasmic tail as palmitoylation sites. Substitution of either cysteine residue by alanine interfered with palmitoylation of the other. Palmitoylation of LPC was found to be sensitive to the protein palmitoyltransferase inhibitor tunicamycin but not cerulenin. It was also insensitive to the drugs brefeldin A, monensin and cycloheximide, indicating that the modification occurs in a late exocytic or endocytic compartment. Turnover of palmitoylated LPC is significantly faster (t1/2 ≈ 50min) than that of the LPC polypeptide backbone (t1/2 ≈ 3h), suggesting that palmitoylation is reversible. Abrogation of palmitoylation reduced the half-life of the LPC protein, but did not affect steady-state localization of LPC in the trans-Golgi network. Finally, LPC could not be detected in detergent-resistant membrane rafts. Taken together, these results suggest that dynamic palmitoylation of LPC is important for stability, but does not function as a dominant trafficking signal.

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Sequences within the Cytoplasmic Domain of Gp180/Carboxypeptidase D Mediate Localization to the Trans-Golgi Network

Molecular Biology of the Cell ◽

10.1091/mbc.10.1.35 ◽

1999 ◽

Vol 10 (1) ◽

pp. 35-46 ◽

Cited By ~ 37

Author(s):

Francis J. Eng ◽

Oleg Varlamov ◽

Lloyd D. Fricker

Keyword(s):

Cell Surface ◽

Secretory Pathway ◽

Point Mutations ◽

Cell Surface Receptor ◽

Type I ◽

Multiple Sequence ◽

Trans Golgi Network ◽

Duck Hepatitis ◽

Surface Receptor ◽

Golgi Network

Gp180, a duck protein that was proposed to be a cell surface receptor for duck hepatitis B virus, is the homolog of metallocarboxypeptidase D, a mammalian protein thought to function in the trans-Golgi network (TGN) in the processing of proteins that transit the secretory pathway. Both gp180 and mammalian metallocarboxypeptidase D are type I integral membrane proteins that contain a 58-residue cytosolic C-terminal tail that is highly conserved between duck and rat. To investigate the regions of the gp180 tail involved with TGN retention and intracellular trafficking, gp180 and various deletion and point mutations were expressed in the AtT-20 mouse pituitary corticotroph cell line. Full length gp180 is enriched in the TGN and also cycles to the cell surface. Truncation of the C-terminal 56 residues of the cytosolic tail eliminates the enrichment in the TGN and the retrieval from the cell surface. Truncation of 12–43 residues of the tail reduced retention in the TGN and greatly accelerated the turnover of the protein. In contrast, deletion of the C-terminal 45 residues, which truncates a potential YxxL-like sequence (FxxL), reduced the protein turnover and caused accumulation of the protein on the cell surface. A point mutation of the FxxL sequence to AxxL slowed internalization, showing that this element is important for retrieval from the cell surface. Mutation of a pair of casein kinase II sites within an acidic cluster showed that they are also important for trafficking. The present study demonstrates that multiple sequence elements within the cytoplasmic tail of gp180 participate in TGN localization.

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Dynamic palmitoylation of lymphoma proprotein convertase prolongs its half-life, but is not essential for trans-Golgi network localization

Biochemical Journal ◽

10.1042/0264-6021:3520827 ◽

2000 ◽

Vol 352 (3) ◽

pp. 827 ◽

Cited By ~ 5

Author(s):

Jan-Willem H.P. van de LOO ◽

Meike TEUCHERT ◽

Ilse PAULI ◽

Evelyn PLETS ◽

Wim J.M. Van de VEN ◽

...

Keyword(s):

Half Life ◽

Proprotein Convertase ◽

Trans Golgi Network ◽

Network Localization ◽

Golgi Network

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Intracellular trafficking of metallocarboxypeptidase D in AtT-20 cells: localization to the trans-Golgi network and recycling from the cell surface

Journal of Cell Science ◽

10.1242/jcs.111.7.877 ◽

1998 ◽

Vol 111 (7) ◽

pp. 877-885 ◽

Cited By ~ 2

Author(s):

O. Varlamov ◽

L.D. Fricker

Keyword(s):

Cell Surface ◽

Wheat Germ Agglutinin ◽

Wheat Germ ◽

Secretory Pathway ◽

Brefeldin A ◽

Full Length ◽

Recycling Endosomes ◽

Trans Golgi Network ◽

Cell Processes ◽

Golgi Network

Carboxypeptidase D (CPD) is a recently discovered membrane-bound metallocarboxypeptidase that has been proposed to be involved in the post-translational processing of peptides and proteins that transit the secretory pathway. In the present study, the intracellular distribution of CPD was examined in AtT-20 cells, a mouse anterior pituitary-derived corticotroph. Antisera to CPD stain the same intracellular structures as those labeled with furin and wheat germ agglutinin. This distribution is distinct from carboxypeptidase E, which is localized to the secretory vesicles in the cell processes. The perinuclear distribution of CPD is detected even when the AtT-20 cells are treated with brefeldin A for 1–30 minutes, suggesting that CPD is present in the trans-Golgi network (TGN). Although CPD is predominantly found in the TGN, an antiserum to the full length protein is internalized within 15–30 minutes of incubation at 37 degrees C. In contrast, an antiserum raised against the C-terminal region of CPD does not become internalized, suggesting that this domain is cytosolic. The antiserum to the full length CPD is internalized to a structure that co-stains with furin and wheat germ agglutinin, but is distinct from transferrin recycling endosomes. The internalization of CPD is not substantially affected by treatment of the AtT-20 cells with brefeldin A. These data are consistent with the cycling of CPD to the cell surface and back to the TGN. The TGN localization of CPD raises the possibility of a role for this enzyme in the processing of proteins that transit the secretory pathway.

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Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targeting of rat lgp120 (lamp-I) in MDCK cells.

The Journal of Cell Biology ◽

10.1083/jcb.128.3.321 ◽

1995 ◽

Vol 128 (3) ◽

pp. 321-332 ◽

Cited By ~ 86

Author(s):

S Höning ◽

W Hunziker

Keyword(s):

Mdck Cells ◽

Transmembrane Protein ◽

Cytoplasmic Tail ◽

Trans Golgi Network ◽

Cytosolic Domain ◽

Lysosomal Sorting ◽

Lysosomal System ◽

Golgi Network ◽

Basolateral Targeting ◽

Cytoplasmic Determinants

Rat lysosomal glycoprotein 120 (lgp120; lamp-I) is a transmembrane protein that is directly delivered from the trans-Golgi network (TGN) to the endosomal/lysosomal system without prior appearance on the cell surface. Its short cytosolic domain of 11 residues encodes determinants for direct lysosomal sorting, endocytosis and, in polarized cells, basolateral targeting. We now characterize the structural requirements in the cytosolic domain required for sorting of lgp120 into the different pathways. Our results show that the cytoplasmic tail is sufficient to mediate direct transport from the trans-Golgi network (TGN) to lysosomes and that a G7-Y8-X-X-I11 motif is crucial for this sorting event. While G7 is only critical for direct lysosomal sorting in the TGN, Y8 and I11 are equally important for lysosomal sorting, endocytosis, and basolateral targeting. Thus, a small motif of five amino acids in the cytoplasmic tail of lgp120 can be recognized by the sorting machinery at several cellular locations and direct the protein into a variety of intracellular pathways.

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The targeting of Lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane.

The Journal of Cell Biology ◽

10.1083/jcb.132.4.565 ◽

1996 ◽

Vol 132 (4) ◽

pp. 565-576 ◽

Cited By ~ 166

Author(s):

J Rohrer ◽

A Schweizer ◽

D Russell ◽

S Kornfeld

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Amino Acid ◽

Cytoplasmic Tail ◽

Kinetic Studies ◽

Type I ◽

Trans Golgi Network ◽

Intracellular Targeting ◽

Sorting Motif ◽

Golgi Network

Lamp1 is a type I transmembrane glycoprotein that is localized primarily in lysosomes and late endosomes. Newly synthesized molecules are mostly transported from the trans-Golgi network directly to endosomes and then to lysosomes. A minor pathway involves transport via the plasma membrane. The 11-amino acid cytoplasmic tail of lamp1 contains a tyrosine-based motif that has been previously shown to mediate sorting in the trans-Golgi network and rapid internalization at the plasma membrane. We studied whether this motif also mediates sorting in endosomes. We found that mutant forms of lamp1 in which all the amino acids of the cytoplasmic tail were modified except for the RKR membrane anchor and the YXXI sorting motif still localized to dense lysosomes, indicating that the YXXI motif is sufficient to confer proper intracellular targeting. However, when the spacing of the YXXI motif relative to the membrane was changed by deleting one amino acid or adding five amino acids, lysosomal targeting was almost completely abolished. Kinetic studies showed that these mutants were trapped in a recycling pathway, involving trafficking between the plasma membrane and early endocytic compartments. These findings indicate that the YXXI signal of lamp1 is recognized at several sorting sites, including the trans-Golgi network, the plasma membrane, and the early/sorting endosomes. Small changes in the spacing of this motif relative to the membrane dramatically impair sorting in the early/sorting endosomes but have only a modest effect on internalization at the plasma membrane. The spacing of sorting signals relative to the membrane may prove to be an important determinant in the functioning of these signals.

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Multimeric connexin interactions prior to the trans-Golgi network

Journal of Cell Science ◽

10.1242/jcs.114.22.4013 ◽

2001 ◽

Vol 114 (22) ◽

pp. 4013-4024

Author(s):

Jayasri Das Sarma ◽

Rita A. Meyer ◽

Fushan Wang ◽

Valsamma Abraham ◽

Cecilia W. Lo ◽

...

Keyword(s):

Brefeldin A ◽

Dominant Negative ◽

Alveolar Epithelial Cells ◽

Trans Golgi Network ◽

Alveolar Epithelial ◽

C Terminus ◽

Gradient Fractionation ◽

Golgi Network ◽

Nih 3T3 ◽

Gap Junction Hemichannels

Cells that express multiple connexins have the capacity to form heteromeric (mixed) gap junction hemichannels. We used a dominant negative connexin construct, consisting of bacterial β-galactosidase fused to the C terminus of connexin43 (Cx43/β-gal), to examine connexin compatibility in NIH 3T3 cells. Cx43/β-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool induced by Cx43/β-gal colocalized with a medial Golgi apparatus marker and was readily disassembled by treatment with brefeldin A. This was unexpected, since previous studies indicated that Cx43 assembly into hexameric hemichannels occurs in the trans-Golgi network (TGN) and is sensitive to brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/β-gal were assembled into a subhexameric complex. Cx43/β-gal also specifically interacted with Cx46, but not Cx32, consistent with the ability of Cx43/β-gal to simultaneously inhibit multiple connexins. We confirmed that interactions between Cx43/β-gal and Cx46 reflect the ability of Cx43 and Cx46 to form heteromeric complexes, using HeLa and alveolar epithelial cells, which express both connexins. In contrast, ROS osteoblastic cells, which differentially sort Cx43 and Cx46, did not form Cx43/Cx46 heteromers. Thus, cells have the capacity to regulate whether or not compatible connexins intermix.

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p230 is associated with vesicles budding from the trans-Golgi network

Journal of Cell Science ◽

10.1242/jcs.109.12.2811 ◽

1996 ◽

Vol 109 (12) ◽

pp. 2811-2821 ◽

Cited By ~ 7

Author(s):

P.A. Gleeson ◽

T.J. Anderson ◽

J.L. Stow ◽

G. Griffiths ◽

B.H. Toh ◽

...

Keyword(s):

Brefeldin A ◽

Immunogold Labeling ◽

Coated Vesicles ◽

Specific Marker ◽

Very High Frequency ◽

Trans Golgi Network ◽

Golgi Membranes ◽

Vesicle Biogenesis ◽

Heptad Repeats ◽

Golgi Network

Transport vesicle formation requires the association of cytosolic proteins with the membrane. We have previously described a brefeldin-A sensitive, hydrophilic protein (p230), containing a very high frequency of heptad repeats, found in the cytosol and associated with Golgi membranes. We show here that p230 is localised on the trans-Golgi network, by immunogold labeling of HeLa cell cryosections using alpha 2,6 sialyltransferase as a compartment-specific marker. The role of G protein activators on the binding of p230 to Golgi membranes and in vesicle biogenesis has been investigated. Treatment of streptolysin-O permeabilised HeLa cells with either GTP gamma S or AlF4- resulted in accumulation of p230 on Golgi membranes. Furthermore, immunolabeling of isolated Golgi membranes treated with AlF4-, to induce the accumulation of vesicles, showed that p230 is predominantly localised to the cytoplasmic surface of trans-Golgi network-derived budding structures and small coated vesicles. p230-labeled vesicles have a thin (approximately 10 nm) electron dense cytoplasmic coat and could be readily distinguished from clathrin-coated vesicles. Dual immunogold labeling of perforated cells, or of cryosections of treated Golgi membranes, revealed that p230 and the trans-Golgi network-associated p200, which we show here to be distinct molecules, appear to be localised on separate populations of vesicles budding from the trans-Golgi network. These results strongly suggest the presence of distinct populations of non-clathrin coated vesicles derived from the trans-Golgi network. As p230 recycles between the cytosol and buds/vesicles of TGN membranes, a process regulated by G proteins, we propose that p230 is involved in the biogenesis of a specific population of non-clathrin coated vesicles.

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The steady state distribution of humTGN46 is not significantly altered in cells defective in clathrin-mediated endocytosis

Journal of Cell Science ◽

10.1242/jcs.111.23.3451 ◽

1998 ◽

Vol 111 (23) ◽

pp. 3451-3458 ◽

Cited By ~ 3

Author(s):

G. Banting ◽

R. Maile ◽

E.P. Roquemore

Keyword(s):

Steady State ◽

Cell Surface ◽

Dominant Negative ◽

Type I ◽

Steady State Distribution ◽

State Distribution ◽

Trans Golgi Network ◽

Defective Mutant ◽

Dynamin I ◽

Golgi Network

It has been shown previously that whilst the rat type I integral membrane protein TGN38 (ratTGN38) is predominantly localised to the trans-Golgi network this protein does reach the cell surface from where it is internalised and delivered back to the trans-Golgi network. This protein thus provides a suitable tool for the investigation of trafficking pathways between the trans-Golgi network and the cell surface and back again. The human orthologue of ratTGN38, humTGN46, behaves in a similar fashion. These proteins are internalised from the cell surface via clathrin mediated endocytosis, a process which is dependent upon the GTPase activity of dynamin. We thus reasoned that humTGN46 would accumulate at the surface of cells rendered defective in clathrin mediated endocytosis by virtue of the fact that they express a GTPase defective mutant of dynamin I. It did not. In fact, expression of a dominant negative GTPase defective mutant of dynamin I had no detectable effect on the steady state distribution of humTGN46. One explanation for this observation is that humTGN46 does not travel directly to the cell surface from the trans-Golgi network. Further studies on cells expressing the dominant negative GTPase defective mutant of dynamin I indicate that the major recycling pathway for humTGN46 is in fact between the trans-Golgi network and the early endosome.

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Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single Golgi compartment

Journal of Cell Science ◽

10.1242/jcs.106.3.815 ◽

1993 ◽

Vol 106 (3) ◽

pp. 815-822

Author(s):

N.J. Bryant ◽

A. Boyd

Keyword(s):

Saccharomyces Cerevisiae ◽

Secretory Pathway ◽

High Yield ◽

Trans Golgi Network ◽

Processing Enzymes ◽

Terminal Domain ◽

Golgi Compartment ◽

Functional Equivalent ◽

Golgi Network

One of the Golgi compartments of Saccharomyces cerevisiae is defined by the presence of a specific endoproteinase, Kex2p, which cleaves precursor polypeptides at pairs of basic residues. We have used antibodies directed against the cytoplasmically disposed C-terminal domain of Kex2p to develop an immuno-affinity procedure for the isolation of Kex2p-containing organelles. The method gives a high yield of sealed organelles that are essentially free of contamination from other secretory pathway organelles while being significantly enriched for two other late Golgi enzymes, dipeptidylaminopeptidase A and the Kex1 carboxypeptidase. Our findings provide clear evidence for a single yeast Golgi compartment containing all three late-processing enzymes, which is likely to be the functional equivalent in yeast of the mammalian trans-Golgi network.

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