Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

1999 ◽  
Vol 112 (11) ◽  
pp. 1721-1732 ◽  
Author(s):  
M.J. Francis ◽  
E.E. Jones ◽  
E.R. Levy ◽  
R.L. Martin ◽  
S. Ponnambalam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Transmembrane Domain ◽  
Menkes Disease ◽  
Cytoplasmic Domain ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Golgi Network

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

scholarly journals The complex interactions of Chs5p, the ChAPs, and the cargo Chs3p

2012 ◽  
Vol 23 (22) ◽  
pp. 4402-4415 ◽  
Author(s):  
Uli Rockenbauch ◽  
Alicja M. Ritz ◽  
Carlos Sacristan ◽  
Cesar Roncero ◽  
Anne Spang
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Chitin Synthase ◽  
Tetratricopeptide Repeats ◽  
Trans Golgi Network ◽  
Complex Interactions ◽  
C Terminus ◽  
Bud Neck ◽  
Domain Switch ◽  
Golgi Network

The exomer complex is a putative vesicle coat required for the direct transport of a subset of cargoes from the trans-Golgi network (TGN) to the plasma membrane. Exomer comprises Chs5p and the ChAPs family of proteins (Chs6p, Bud7p, Bch1p, and Bch2p), which are believed to act as cargo receptors. In particular, Chs6p is required for the transport of the chitin synthase Chs3p to the bud neck. However, how the ChAPs associate with Chs5p and recognize cargo is not well understood. Using domain-switch chimeras of Chs6p and Bch2p, we show that four tetratricopeptide repeats (TPRs) are involved in interaction with Chs5p. Because these roles are conserved among the ChAPs, the TPRs are interchangeable among different ChAP proteins. In contrast, the N-terminal and the central parts of the ChAPs contribute to cargo specificity. Although the entire N-terminal domain of Chs6p is required for Chs3p export at all cell cycle stages, the central part seems to predominantly favor Chs3p export in small-budded cells. The cargo Chs3p probably also uses a complex motif for the interaction with Chs6, as the C-terminus of Chs3p interacts with Chs6p and is necessary, but not sufficient, for TGN export.

scholarly journals Yeast P4-ATPases Drs2p and Dnf1p Are Essential Cargos of the NPFXD/Sla1p Endocytic Pathway

2007 ◽  
Vol 18 (2) ◽  
pp. 487-500 ◽  
Author(s):  
Ke Liu ◽  
Zhaolin Hua ◽  
Joshua A. Nepute ◽  
Todd R. Graham
Keyword(s):  
Plasma Membrane ◽  
Protein Transport ◽  
Endocytic Pathway ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Sensitive Mutant ◽  
C Terminus ◽  
P Type ◽  
Golgi Network ◽  
Endocytic Pathways

Drs2p family P-type ATPases (P4-ATPases) are required in multiple vesicle-mediated protein transport steps and are proposed to be phospholipid translocases (flippases). The P4-ATPases Drs2p and Dnf1p cycle between the exocytic and endocytic pathways, and here we define endocytosis signals required by these proteins to maintain a steady-state localization to internal organelles. Internalization of Dnf1p from the plasma membrane uses an NPFXD endocytosis signal and its recognition by Sla1p, part of an endocytic coat/adaptor complex with clathrin, Pan1p, Sla2p/End4p, and End3p. Drs2p has multiple endocytosis signals, including two NPFXDs near the C terminus and PEST-like sequences near the N terminus that may mediate ubiquitin (Ub)-dependent endocytosis. Drs2p localizes to the trans-Golgi network in wild-type cells and accumulates on the plasma membrane when both the Ub- and NPFXD-dependent endocytic mechanisms are inactivated. Surprisingly, the pan1-20 temperature-sensitive mutant is constitutively defective for Ub-dependent endocytosis but is not defective for NPFXD-dependent endocytosis at the permissive growth temperature. To sustain viability of pan1-20, Drs2p must be endocytosed through the NPFXD/Sla1p pathway. Thus, Drs2p is an essential endocytic cargo in cells compromised for Ub-dependent endocytosis. These results demonstrate an essential role for endocytosis in retrieving proteins back to the Golgi, and they define critical cargos of the NPFXD/Sla1p system.

scholarly journals Estimation of the amount of internalized ricin that reaches the trans-Golgi network

1988 ◽  
Vol 106 (2) ◽  
pp. 253-267 ◽  
Author(s):  
B van Deurs ◽  
K Sandvig ◽  
OW Petersen ◽  
S Olsnes ◽  
K Simons ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Golgi Complex ◽  
Endocytic Pathway ◽  
Membrane Glycoproteins ◽  
Double Labeling ◽  
Trans Golgi Network ◽  
A Chain ◽  
Golgi Network ◽  
Ricin A

We have used a protocol for internalization of ricin, a ligand binding to plasma membrane glycoproteins and glycolipids with terminal galactosyl residues, and infection with the vesicular stomatitis virus ts 045 mutant in BHK-21 cells to determine whether internalized plasma membrane molecules tagged by ricin reach distinct compartments of the biosynthetic-exocytic pathway. At 39.5 degrees C newly synthesized G protein of ts 045 was largely prevented from leaving the endoplasmic reticulum. At the same temperature ricin was endocytosed and reached, in addition to endosomes and lysosomes, elements of the Golgi complex. When the temperature was lowered to 19.5 degrees C, no more ricin was delivered to the Golgi complex, but now G protein accumulated in the Golgi stacks and the trans-Golgi network (TGN). Double-labeling immunogold cytochemistry on ultracryosections was used to detect G protein and ricin simultaneously. These data, combined with stereological and biochemical methods, showed that approximately 5% of the total amount of ricin within the cells, corresponding to 6-8 X 10(4) molecules per cell, colocalized with G protein in the Golgi complex after 60 min at 39.5 degrees C. Of this amount approximately 70-80% was present in the TGN. Since most of the ricin molecules remain bound to their binding sites at the low pH prevailing in compartments of the endocytic pathway, the results indicate that a fraction of the internalized plasma membrane molecules with terminal galactose are not recycled directly from endosomes or delivered to lysosomes, but are routed to the Golgi complex. Also, the results presented here, in combination with other recent studies on ricin internalization, suggest that translocation of the toxic ricin A-chain to the cytosol occurs in the TGN.

The N-terminal cysteine cluster is essential for membrane targeting of B/K protein

2001 ◽  
Vol 360 (2) ◽  
pp. 441-448 ◽  
Author(s):  
Mitsunori FUKUDA ◽  
Katsuhiko MIKOSHIBA
Keyword(s):  
Transmembrane Domain ◽  
Protein Motif ◽  
Binding Motifs ◽  
Trans Golgi Network ◽  
Terminal Domain ◽  
C Terminus ◽  
Type C ◽  
Golgi Network ◽  
Membrane Anchoring ◽  
Specific Ligand

B/K protein belongs to a family of C-terminal-type (C-type) tandem C2 proteins that contain two C2 Ca2+-binding motifs at the C-terminus. Although other C-type tandem C2 proteins have been found to have a unique N-terminal domain that is involved in membrane anchoring (e.g. synaptotagmin) or specific ligand binding (e.g. rabphilin-3A and Doc2), no research has been conducted on the function of the N-terminal domain of B/K protein. In this study we showed that despite lacking a transmembrane domain, both native and recombinant B/K proteins are tightly bound to the membrane fraction, which was completely resistant to 0.1M Na2CO3, pH11, or 1M NaCl treatment. Deletion and mutation analyses indicated that the cysteine cluster at the N-terminal domain (consisting of seven cysteine residues, Cys-19, Cys-23, Cys-26, Cys-27, Cys-30, Cys-35 and Cys-36) is essential for the membrane localization of B/K protein. When wild-type B/K was expressed in PC12 cells, B/K proteins were localized mainly in the perinuclear region (trans-Golgi network), whereas mutant B/K proteins carrying Cys-to-Ala substitutions were present in the cytosol. Based on our findings, we propose that the N-terminal domain of B/K protein contains a novel cysteine-based protein motif that may allow B/K protein to localize in the trans-Golgi network.

scholarly journals Nanoscopical analysis reveals an orderly arrangement of the presynaptic scaffold protein Bassoon at the Golgi-apparatus

2021 ◽  
Author(s):  
Tina Ghelani ◽  
Carolina Montenegro ◽  
Anna Fejtova ◽  
Thomas Dresbach
Keyword(s):  
Golgi Apparatus ◽  
Active Zone ◽  
Hippocampal Neurons ◽  
Scaffold Protein ◽  
Stimulated Emission ◽  
Trans Golgi Network ◽  
C Terminus ◽  
N Terminus ◽  
Orderly Fashion ◽  
Golgi Network

Bassoon is a core scaffold protein of the presynaptic active zone. In brain synapses, the C-terminus of Bassoon is oriented toward the plasma membrane and its N-terminus is oriented towards synaptic vesicles. At the Golgi-apparatus Bassoon is thought to assemble active zone precursor structures, but whether it is arranged in an orderly fashion is unknown. Understanding the topology of this large scaffold protein is important for models of active zone biogenesis. Using stimulated emission depletion nanoscopy in cultured hippocampal neurons, we found that an N-terminal intramolecular tag of recombinant Bassoon, but not C-terminal tag, colocalized with markers of the trans-Golgi network. The N-terminus of Bassoon was located between 48 nm and 69 nm away from TGN38, while its C-terminus was located between 100 nm and 115 nm away from TGN38. Sequences within the first 95 amino acids of Bassoon were required for this arrangement. Our data are consistent with a model, in which the N-terminus of Bassoon binds to the membranes of the trans-Golgi network, while the C-terminus associates with active zone components, thus reflecting the topographic arrangement characteristic of synapses also at the Golgi-apparatus.

scholarly journals Exogenous MAL Reroutes Selected Hepatic Apical Proteins into the Direct Pathway in WIF-B Cells

2007 ◽  
Vol 18 (7) ◽  
pp. 2707-2715 ◽  
Author(s):  
Sai Prasad Ramnarayanan ◽  
Christina A. Cheng ◽  
Maria Bastaki ◽  
Pamela L. Tuma
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Epithelial Cells ◽  
Transmembrane Domain ◽  
Apical Membrane ◽  
Apical Plasma Membrane ◽  
Indirect Pathway ◽  
Trans Golgi Network ◽  
Direct Pathway ◽  
Golgi Network

Unlike simple epithelial cells that directly target newly synthesized glycophosphatidylinositol (GPI)-anchored and single transmembrane domain (TMD) proteins from the trans-Golgi network to the apical membrane, hepatocytes use an indirect pathway: proteins are delivered to the basolateral domain and then selectively internalized and transcytosed to the apical plasma membrane. Myelin and lymphocyte protein (MAL) and MAL2 have been identified as regulators of direct and indirect apical delivery, respectively. Hepatocytes lack endogenous MAL consistent with the absence of direct apical targeting. Does MAL expression reroute hepatic apical residents into the direct pathway? We found that MAL expression in WIF-B cells induced the formation of cholesterol and glycosphingolipid-enriched Golgi domains that contained GPI-anchored and single TMD apical proteins; polymeric IgA receptor (pIgA-R), polytopic apical, and basolateral resident distributions were excluded. Basolateral delivery of newly synthesized apical residents was decreased in MAL-expressing cells concomitant with increased apical delivery; pIgA-R and basolateral resident delivery was unchanged. These data suggest that MAL rerouted selected hepatic apical proteins into the direct pathway.

scholarly journals Structural Determinants Critical for Localization and Signaling within the Seventh Transmembrane Domain of the Type 1 Corticotropin Releasing Hormone Receptor: Lessons from the Receptor Variant R1d

2008 ◽  
Vol 22 (11) ◽  
pp. 2505-2519 ◽  
Author(s):  
Danijela Markovic ◽  
Hendrik Lehnert ◽  
Michael A. Levine ◽  
Dimitris K. Grammatopoulos
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Receptor Expression ◽  
Site Directed Mutagenesis ◽  
Functional Responses ◽  
Membrane Expression ◽  
Recombinant Receptors ◽  
C Terminus

Abstract The type 1 CRH receptor (CRH-R1) plays a fundamental role in homeostatic adaptation to stressful stimuli. CRH-R1 gene activity is regulated through alternative splicing and generation of various CRH-R1 mRNA variants. One such variant is the CRH-R1d, which has 14 amino acids missing from the putative seventh transmembrane domain due to exon 13 deletion, a splicing event common to other members of the B1 family of G protein-coupled receptors. In this study, using overexpression of recombinant receptors in human embryonic kidney 293 and myometrial cells, we showed by confocal microscopy that in contrast to CRH-R1α, the R1d variant is primarily retained in the cytoplasm, although some cell membrane expression is also evident. Use of antibodies against the CRH-R1 C terminus in nonpermeabilized cells showed that membrane-expressed CRH-R1d contains an extracellular C terminus. Interestingly, treatment of CRH-R1d-expressing cells with CRH (100 nM) for 45–60 min elicited functional responses associated with a significant reduction of plasma membrane receptor expression, redistribution of intracellular receptors, and increased receptor degradation. Site-directed mutagenesis studies identified the cassette G356-F358 within transmembrane domain 7 as crucial for CRH-R1α stability to the plasma membrane because deletion of this cassette caused substantial intracellular localization of CRH-R1 α. Most importantly, coexpression studies between CRH-R1d and CRH-R2β demonstrated that the CRH-R2β could partially rescue CRH-R1d membrane expression, and this was associated with a significant attenuation of urocotrin II-induced cAMP production and ERK1/2 and p38MAPK activation, suggesting that CRH-R1d might specifically induce heterologous impairment of CRH-R2 signaling responses. This mechanism appears to involve accelerated CRH-R2β endocytosis.

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.

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