Endosome to trans-Golgi network transport of Proprotein Convertase 7 is mediated by a cluster of basic amino acids and palmitoylated cysteines

The Golgi apparatus (GA) is the organelle where complex glycan formation takes place. In addition, it is a major sorting site for proteins destined for various subcellular compartments or for secretion. Here we investigate β1,4-galactosyltransferase 1 (galT) and α2,6-sialyltransferase 1 (siaT), two trans-Golgi glycosyltransferases, with respect to their different pathways in monensin-treated cells. Upon addition of monensin galT dissociates from siaT and the GA and accumulates in swollen vesicles derived from the trans-Golgi network (TGN), as shown by colocalization with TGN46, a specific TGN marker. We analyzed various chimeric constructs of galT and siaT by confocal fluorescence microscopy and time-lapse videomicroscopy as well as Optiprep density gradient fractionation. We show that the first 13 amino acids of the cytoplasmic tail of galT are necessary for its localization to swollen vesicles induced by monensin. We also show that the monensin sensitivity resulting from the cytoplasmic tail can be conferred to siaT, which leads to the rapid accumulation of the galT–siaT chimera in swollen vesicles upon monensin treatment. On the basis of these data, we suggest that cycling between the trans-Golgi cisterna and the trans-Golgi network of galT is signal mediated.

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Active PIKfyve Associates with and Promotes the Membrane Attachment of the Late Endosome-to-trans-Golgi Network Transport Factor Rab9 Effector p40

Journal of Biological Chemistry ◽

10.1074/jbc.m307260200 ◽

2003 ◽

Vol 278 (51) ◽

pp. 50863-50871 ◽

Cited By ~ 48

Author(s):

Ognian C. Ikonomov ◽

Diego Sbrissa ◽

Krzysztof Mlak ◽

Robert Deeb ◽

Jason Fligger ◽

...

Keyword(s):

Late Endosome ◽

Trans Golgi Network ◽

Network Transport ◽

Transport Factor ◽

Membrane Attachment ◽

Golgi Network

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

Biochemical Journal ◽

10.1042/bj3520827 ◽

2000 ◽

Vol 352 (3) ◽

pp. 827-833 ◽

Cited By ~ 15

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 ◽

Secretory Pathway ◽

Transmembrane Protein ◽

Brefeldin A ◽

Cytoplasmic Tail ◽

Cysteine Residue ◽

Type I ◽

Proprotein Convertase ◽

Trans Golgi Network ◽

Golgi Network

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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Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin.

The EMBO Journal ◽

10.1002/j.1460-2075.1995.tb07240.x ◽

1995 ◽

Vol 14 (11) ◽

pp. 2424-2435 ◽

Cited By ~ 171

Author(s):

W. Schäfer ◽

A. Stroh ◽

S. Berghöfer ◽

J. Seiler ◽

M. Vey ◽

...

Keyword(s):

Cytoplasmic Domain ◽

Endosomal Trafficking ◽

Proprotein Convertase ◽

Trans Golgi Network ◽

Network Localization ◽

Targeting Signals ◽

Golgi Network

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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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