Targeting of membrane proteins to endosomes and lysosomes (original) (raw)
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Delivery of endocytosed membrane proteins to the lysosome
Biochimica Et Biophysica Acta-molecular Cell Research, 2009
The delivery of proteins from the plasma membrane to the lysosome for degradation is essential for normal cellular function. There is now a good understanding of the protein complexes involved in sorting proteins at the plasma membrane and into the intralumenal vesicles of the multi-vesicular body. A combination of cell free content mixing assays and live-cell imaging has dissected out the final step in delivery of macromolecules to the lysosome from the multi-vesicular body and provided insights into the molecular mechanisms by which late endosomes and lysosomes exchange lumenal contents. The endocytic pathway has provided a platform with which to understand the autophagic and phagocytic pathways, but the fine details of how traffic through these pathways is regulated remain to be determined.
Bidirectional traffic between the Golgi and the endosomes – machineries and regulation
Journal of Cell Science, 2016
The bidirectional transport between the Golgi complex and the endocytic pathway has to be finely regulated in order to ensure the proper delivery of newly synthetized lysosomal enzymes and the return of sorting receptors from degradative compartments. The high complexity of these routes has led to experimental difficulties in properly dissecting and separating the different pathways. As a consequence, several models have been proposed during the past decades. However, recent advances in our understanding of endosomal dynamics have helped to unify these different views. We provide here an overview of the current insights into the transport routes between Golgi and endosomes in mammalian cells. The focus of the Commentary is on the key molecules involved in the trafficking pathways between these intracellular compartments, such as Rab proteins and sorting receptors, and their regulation. A proper understanding of the bidirectional traffic between the Golgi complex and the endolysosomal system is of uttermost importance, as several studies have demonstrated that mutations in the factors involved in these transport pathways result in various pathologies, in particular lysosome-associated diseases and diverse neurological disorders, such as Alzheimer's and Parkinson's disease.
The Golgi-targeting sequence of the peripheral membrane protein p230
Journal of Cell Science, 1999
Vesicle transport requires the recruitment of cytosolic proteins to specific membrane compartments. We have previously characterised a brefeldin A-sensitive trans-Golgi network-localised protein (p230) that is associated with a population of non-clathrin-coated vesicles. p230 recycles between the cytosol and the cytoplasmic face of buds/vesicles of trans-Golgi network membranes in a G protein-regulated manner. Identifying the mechanism responsible for Golgi targeting of p230 is important for the elucidation of its function. By transfection of COS cells with deletion mutants of p230 we here demonstrate that the C-terminal domain is necessary for targeting to the Golgi. Furthermore, the C-terminal 98 amino acid domain of p230 attached to the green fluorescent protein (GFP-p230-C98aa) was efficiently Golgi-localised in transfected COS cells. Deletion mutants of GFP-p230-C98aa together with alanine scanning mutagenesis identified a minimum stretch of 42 amino acids that is essential for...
1992
The 100-110-kD proteins (c~-, #-, B'-, and 7-adaptins) of clathrin-coated vesicles and the 110-kD protein (B-COP) of the nonclathrin-coated vesicles that mediate constitutive transport through the Golgi have homologous protein sequences. To determine whether homologous processes are involved in assembly of the two types of coated vesicles, the membrane binding properties of their coat proteins were compared. After treatment of MDBK cells with the fungal metabolite Brefeldin A (BFA), ~ff-COP was redistributed to the cytoplasm within 15 s, 3,-adaptin and clathrin in the trans-Golgi network (TGN) dispersed within 30 s, but the ot-adaptin and clathrin present on coated pits and vesicles derived from the plasma membrane remained membrane associated even after a 15-min exposure to BFA. In PtKI cells and MDCK cells, BFA did not affect/~-COP binding or Golgi morphology but still induced redistribution of 7-adaptin and clathrin from TGN membranes to the cytoplasm. Thus BFA affects the binding of coat proteins to membranes in the Golgi region (Golgi apparatus and TGN) but not plasma membranes. However, the Golgi binding interactions of B-COP and 7-adaptin are distinct and differentially sensitive to BFA. BFA treatment did not release 7-adaptin or clathrin from purified clathrin-coated vesicles, suggesting that their distribution to the cytoplasm after BFA treatment of cells was due to interference with their rebinding to TGN membranes after a normal cycle of disassembly. This was confirmed using an in vitro assay in which 7-adaptin binding to TGN membranes was blocked by BFA and enhanced by GTPTS, similar to the binding of/~-COP to Golgi membranes. These results suggest the involvement of GTP-dependent proteins in the association of the 100-kD coat proteins with membranes in the Golgi region of the cell. p ROTEIN transport between membrane-bound cellular compartments is mediated by protein-coated vesicular carriers. Clathrin-coated vesicles are responsible for selective budding of proteins during receptor-mediated endocytosis and the sorting of proteins in the trans-Golgi network (TGN)~ (6, 29). The coat of the vesicles that mediate nonselective, constitutive export of proteins through the Golgi apparatus to the plasma membrane is composed of COP (coat protein) subunits of 160-, 110-, 98-and 61-kD (23, 35). The 100-kD coat components ofclathrin-coated and COP-coated vesicles have homologous protein sequences and domain structures, suggesting that assembly of these vesicular coats may share some biochemical properties. In clathrin-coated vesicles, the 100-kD proteins are subunits of multimeric protein complexes called adaptors (29) or assembly polypeptides (16). Adaptors bind clathrin and recognize a specific motif in the cytoplasmic tails of receptors (28, 37), thereby linking the clathrin coat to the vesicle membrane and allowing receptors to be sequestered and
hVps41 and VAMP7 function in direct TGN to late endosome transport of lysosomal membrane proteins
Nature communications, 2013
Targeted delivery of lysosome-associated membrane proteins is important for lysosome stability and function. Here we identify a pathway for transport of lysosome-associated membrane proteins directly from the trans-Golgi network to late endosomes, which exists in parallel to mannose 6-phosphate receptor and clathrin-dependent transport of lysosomal enzymes to early endosomes. By immunoelectron microscopy we localized endogenous LAMP-1 and -2 as well as LAMP-1-mGFP to non-coated, biosynthetic carriers at the trans-Golgi network and near late endosomes. These LAMP carriers were negative for mannose 6-phosphate receptor, adaptor-protein complex-1, secretory albumin and endocytic markers, but contained the homotypic fusion and protein sorting complex component hVps41 and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein VAMP7. Knockdown of hVps41 or VAMP7 resulted in the accumulation of lysosome-associated membrane protein carriers, whereas knockdown of hVps39 or hVps18 did not, indicating that the effect of hVps41 is independent of CORVET/HOPS. Mannose 6-phosphate receptor carriers remained unaffected upon hVps41 or VAMP7 knockdown, implicating that hVps41 and VAMP7 are specifically involved in the fusion of trans-Golgi network-derived lysosome-associated membrane protein carriers with late endosomes.
Molecular biology of the cell, 1998
TGN38 is one of the few known resident integral membrane proteins of the trans-Golgi network (TGN). Since it cycles constitutively between the TGN and the plasma membrane, TGN38 is ideally suited as a model protein for the identification of post-Golgi trafficking motifs. Several studies, employing chimeric constructs to detect such motifs within the cytosolic domain of TGN38, have identified the sequence 333YQRL336 as an autonomous signal capable of localizing reporter proteins to the TGN. In addition, one group has found that an upstream serine residue, S331, may also play a role in TGN38 localization. However, the nature and degree of participation of S331 in the localization of TGN38 remain uncertain, and the effect has been studied in chimeric constructs only. Here we investigate the role of S331 in the context of full-length TGN38. Mutations that abolish the hydroxyl moiety at position 331 (A, D, and E) lead to missorting of endocytosed TGN38 to the lysosome. Conversely, mutati...
Exit from the trans-Golgi network: from molecules to mechanisms
Current Opinion in Cell Biology, 2011
The trans-Golgi network is a major sorting platform of the secretory pathway from which proteins and lipids, both newly synthesized and retrieved from endocytic compartments, are targeted to different destinations. These sorting processes occur during the formation of pleomorphic tubular-vesicular carriers. The past years have provided insights into basic mechanisms coordinating the spatial and temporal organization of machineries necessary for the segregation of membrane components into distinct microdomains, for the bending, elongation, and fission of corresponding membranes, thus revealing a complex interplay of protein-protein and protein-lipid interactions. SL: Cargo-and adaptor-specific mechanisms regulate clathrin-mediated endocytosis. J Cell Biol 2010, 188:919-933. This study shows that the size and turn-over of endocytic clathrin-coated carriers depend on cargo content. 25. Kirchhausen T: Imaging endocytic clathrin structures in living cells. Trends Cell Biol 2009, 19:596-605. 26. Legendre-Guillemin V, Wasiak S, Hussain NK, Angers A, McPherson PS: ENTH/ANTH proteins and clathrin-mediated membrane budding.
Molecular Biology of the Cell, 2004
The precise cellular function of Arl1 and its effectors, the GRIP domain Golgins, is not resolved, despite our recent understanding that Arl1 regulates the membrane recruitment of these Golgins. In this report, we describe our functional study of Golgin-97. Using a Shiga toxin B fragment (STxB)-based in vitro transport assay, we demonstrated that Golgin-97 plays a role in transport from the endosome to the trans-Golgi network (TGN). The recombinant GRIP domain of Golgin-97 as well as antibodies against Golgin-97 inhibited the transport of STxB in vitro. Membrane-associated Golgin-97, but not its cytosolic pool, was required in the in vitro transport assay.