TGN38/41 recycles between the cell surface and the TGN: brefeldin A affects its rate of return to the TGN.

TGN38 and TGN41 are isoforms of an integral membrane protein (TGN38/41) that is predominantly localized to the trans-Golgi network (TGN) of normal rat kidney cells. Polyclonal antisera to TGN38/41 have been used to monitor its appearance at, and removal from, the surface of control and Brefeldin A (BFA)-treated cells. Antibodies that recognize the lumenal domain of TGN38/41 are capable of specific binding to the surface of both control and BFA-treated cells. In both control and BFA-treated cells internalized TGN38/41 is targeted to the TGN; however, there are differences in 1) the morphology of the intracellular structures through which TGN38/41 passes and 2) the kinetics of internalization. These data demonstrate that TGN38/41 cycles between the plasma membrane and the TGN in control and BFA-treated cells and suggest that recycling pathways between the plasma membrane and the TGN exist for predominantly TGN proteins as well as those that normally cycle to other intracellular compartments. They also demonstrate that addition of BFA not only alters the morphology and localization of the TGN but also the kinetics of endocytosis.     

Epitope mapping of two isoforms of a trans Golgi network specific integral membrane protein TGN38/41.

TGN38/41 is an integral membrane protein predominantly located in the trans Golgi network (TGN) of rat (NRK) cells. We have used a cDNA expression system to map the epitopes recognised by a panel of antibodies raised to TGN38/41 as a preliminary step in the accurate identification of the region(s) of the molecule responsible for its correct intracellular location. These studies have confirmed the predicted topology of the molecule, and have identified a region in the cytoplasmic domain which is immunologically (and hence potentially functionally) conserved between species.     

Direct interaction of the trans-Golgi network membrane protein, TGN38, with the F-actin binding protein, neurabin.

TGN38 is a type I integral membrane protein that constitutively cycles between the trans-Golgi network (TGN) and plasma membrane. The cytosolic domain of TGN38 interacts with AP2 clathrin adaptor complexes via the tyrosine-containing motif (-SDYQRL-) to direct internalization from the plasma membrane. This motif has previously been shown to direct both internalization and subsequent TGN targeting of TGN38. We have used the cytosolic domain of TGN38 in a two-hybrid screen, and we have identified the brain-specific F-actin binding protein neurabin-I as a TGN38-binding protein. We demonstrate a direct interaction between TGN38 and the ubiquitous homologue of neurabin-I, neurabin-II (also called spinophilin). We have used a combination of yeast two-hybrid and in vitro protein interaction assays to show that this interaction is dependent on the serine (but not tyrosine) residue of the known TGN38 trafficking motif. We show that TGN38 interacts with the coiled coil region of neurabin in vitro and binds preferentially with the dimeric form of neurabin. TGN38 and neurabin also interact in vivo as demonstrated by coimmunoprecipitation from stably transfected PC12 cells. These data suggest that neurabin provides a direct physical link between TGN38-containing membranes and the actin cytoskeleton.     

An Endocytosed TGN38 Chimeric Protein Is Delivered to the TGN after Trafficking through the Endocytic Recycling Compartment in CHO Cells

To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and ¹²⁵I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38''s postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38 is on the plasma membrane. Kinetic analysis of trafficking of antibodies bound to TacTGN38 showed that after short endocytic pulses, 80% of internalized anti-Tac returned to the cell surface (t _1/2 = 9 min), and the remainder trafficked to the TGN. When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t _1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t _1/2. Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.     

Transferrin receptor containing the SDYQRL motif of TGN38 causes a reorganization of the recycling compartment but is not targeted to the TGN

The SDYQRL motif of the cytoplasmic domain of TGN38 is involved in targeting TGN38 from endosomes to the TGN. To create a system for studying this pathway, we replaced the native transferrin receptor (TR) internalization motif (YTRF) with the SDYQRL TGN-targeting motif. The advantages of using TR as a reporter molecule include the ability to monitor trafficking, in both biochemical and microscopy experiments, using the natural ligand transferrin. When expressed in CHO cells, the SDYQRL-TR construct accumulated in juxtanuclear tubules and vesicles that are in the vicinity of the TGN. The SDYQRL-TR-containing structures, however, do not colocalize with TGN markers (e.g., NBD ceramide), and therefore the SDYQRL motif is not sufficient to target the TR to the TGN. The morphology of the SDYQRL-TR-containing juxtanuclear structures is different from the recycling compartment found in cells expressing the wild-type TR. In addition, the SDYQRL-TR- containing juxtanuclear compartment is more acidic than the recycling compartment in cells expressing the wild-type TR. The juxtanuclear compartment, however, is a bona fide recycling compartment since SDYQRL- TR was recycled back to the cell surface at a rate comparable to the wild-type TR, and sphingomyelin and cellubrevin, both of which label all compartments of the endocytic recycling pathway, colocalize with SDYQRL-TR in the juxtanuclear structures. These findings demonstrate that expression of the SDYQRL-TR construct alters the morphology and pH of endocytic recycling compartments rather than selectively affecting the intracellular trafficking pathway of the SDYQRL-TR construct. Therefore, the SDYQRL trafficking motif is not simply a molecular address that targets proteins to the TGN, but it can play an active role in determining the physical characteristics of endosomal compartments.     

TGN38/41: a molecule on the move

TGN38/41 is a heterodimeric integral membrane protein that cycles between the trans Golgi network and the cell surface. A tyrosine-containing tetrapeptide motif within its cytoplasmic tail is necessary and sufficient for determining its steady-state location in the TGN. Recent results have shown that TGN38/41 plays an essential role in the formation of exocytic vesicles at the TGN by serving as a receptor for complexes of a cytoplasmic protein known as p62, and one of four small GTP-binding proteins, including rab6. For budding to occur, this complex must bind to the cytoplasmic domain of TGN38/41. We propose here that TGN38/41 may couple the segregation of secretory proteins to the budding of exocytic vesicles at the TGN.     

TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain.

Sorting of proteins destined for different plasma membrane domains, lysosomes and secretory pathways takes place in the trans-Golgi network (TGN). TGN38 is an integral membrane protein found in this intracellular compartment. We show that TGN38 contains an autonomous targeting signal within its cytoplasmic domain which determines its intracellular location. Deletion analysis and site-directed mutagenesis of this domain demonstrate that a tyrosine motif homologous to the internalization signal of surface receptors is necessary and sufficient for correct localization. These findings suggest that TGN38 is maintained in the TGN by retrieval from the plasma membrane and employs a different mechanism for retention from that of the transferase enzymes of the trans-Golgi.     

Retrieval of TGN proteins from the cell surface requires endosomal acidification.

TGN38 is a protein of unknown function located in the trans-Golgi network (TGN) of mammalian cells. Its intracellular distribution is maintained by it being continuously retrieved from the plasma membrane. In this paper we show that when cells are treated with agents such as chloroquine which neutralize acidic organelles, the movement of TGN38 along the endocytic pathway is blocked. The same effect is observed with a second TGN protein, the protease furin. We show that the cytoplasmic tail of furin is sufficient to confer a chloroquine-sensitive TGN localization on a heterologous protein. These results imply that the internal pH of endosomes affects sorting processes mediated by signals in the cytoplasmic portion of proteins and have implications for the role of acidification in endosomal function.     

Chimeric forms of furin and TGN38 are transported with the plasma membrane in the trans-Golgi network via distinct endosomal pathways.

Furin and TGN38 are menbrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N.,W.G. Mallet,T.T. Soe,T.E.McGraw, and F.R. Maxfield.1998.J.Cell Biol.142:923-936).Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independentpathways for endosomal transport of proteins to the TGN from the plasma membrane.     

TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.     

Efficient Trafficking of TGN38 from the Endosome to the trans-Golgi Network Requires a Free Hydroxyl Group at Position 331 in the Cytosolic Domain

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, mutation of S331 to T has little effect on the endocytic trafficking of TGN38. Together, these findings indicate that the S331 hydroxyl group has a direct or indirect effect on the ability of the cytosolic tail of TGN38 to interact with trafficking and/or sorting machinery at the level of the early endosome. In addition, mutation of S331 to either A or D results in increased levels of TGN38 at the cell surface. The results confirm that S331 plays a critical role in the intracellular trafficking of TGN38 and further reveal that TGN38 undergoes a signal-mediated trafficking step at the level of the endosome.     

The luminal domain of TGN38 interacts with integrin beta 1 and is involved in its trafficking

TGN38 luminal domain (TGN38LD) was expressed in Cos-7 cells to identify potential binding partners. The luminal domain was secreted but, surprisingly, a significant portion bound to the plasma membrane. Cells over-expressing TGN38LD or the full-length molecule detached from the substratum and left footprints positive for TGN38. Unexpectedly, in these cells, TGN38 colocalizes with integrin α5β1 at the Golgi, the cell surface or in the footprints and an increased amount of both integrin subunits on the plasma membrane was observed. Under physiological conditions when TGN38 is not overexpressed, it interacts with integrin β1. This was demonstrated by reciprocal co-immunoprecipitation of integrin β1 and TGN38. Functional analysis reveals that modification of the trafficking of TGN38 results in a parallel change in the distribution of integrin α5β1, leading to the conclusion that TGN38 is involved in the trafficking of integrin β1.     

Rapid Movement of Newly Synthesized Chicken Apolipoprotein AI to Trans-Golgi Network and Its Secretion in Madin–Darby Canine Kidney Cells

MDCK cells were utilized to study the biosynthesis and secretion of chicken apolipoprotein AI (apoAI). A full-length apoAI cDNA in an eukaryotic expression vector was transfected into a MDCK-TGG cell line, expressing a trans-Golgi network (TGN) marker (TGG), and stable clones expressing apoAI were selected. Pulse–chase and cell fractionation studies showed that, compared to gp 80 (an endogenous secretory protein), apoAI was rapidly transported from RER to Golgi complex within 5 min and released from the Golgi complex to the cell exterior by 30 min. Immunofluorescence and three-dimensional laser scanning confocal microscopy revealed that at steady state apoAI was predominantly localized in the TGN. Transferrin uptake experiments showed that apoAI, localized in the TGN, was derived primarily from biosynthetic and not from endocytic routes. ApoAI was secreted in a nonpolarized manner. We suggest that apoAI stays transiently in the TGN prior to its secretion and that the major events of apoAI biosynthesisin vivoand in MDCK cells are conserved. Possible mechanisms of rapid ER to Golgi transport and TGN localization of apoAI are discussed.     

Lumenal and Transmembrane Domains Play a Role in Sorting Type I Membrane Proteins on Endocytic Pathways

Previous studies have shown that when the cytosolic domains of the type I membrane proteins TGN38 and lysosomal glycoprotein 120 (lgp120) are added to a variety of reporter molecules, the resultant chimeric molecules are localized to the trans-Golgi network (TGN) and to lysosomes, respectively. In the present study we expressed chimeric constructs of rat TGN38 and rat lgp120 in HeLa cells. We found that targeting information in the cytosolic domain of TGN38 could be overridden by the presence of the lumenal and transmembrane domains of lgp120. In contrast, the presence of the transmembrane and cytosolic domains of TGN38 was sufficient to deliver the lumenal domain of lgp120 to the trans-Golgi network. On the basis of steady-state localization of the various chimeras and antibody uptake experiments, we propose that there is a hierarchy of targeting information in each molecule contributing to sorting within the endocytic pathway. The lumenal and cytosolic domains of lgp120 contribute to sorting and delivery to lysosomes, whereas the transmembrane and cytosolic domains of TGN38 contribute to sorting and delivery to the trans-Golgi network.     

BFA‐induced compartments from the Golgi apparatus and trans‐Golgi network/early endosome are distinct in plant cells

Brefeldin A (BFA) is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. At low concentrations (5–10 μg ml^?1), BFA caused both the Golgi apparatus and trans‐Golgi network (TGN), an early endosome (EE) equivalent in plant cells, to form visible aggregates in transgenic tobacco BY‐2 cells. Here we show that these BFA‐induced aggregates from the Golgi apparatus and TGN are morphologically and functionally distinct in plant cells. Confocal immunofluorescent and immunogold electron microscope (EM) studies demonstrated that BFA‐induced Golgi‐ and TGN‐derived aggregates are physically distinct from each other. In addition, the internalized endosomal marker FM4‐64 co‐localized with the TGN‐derived aggregates but not with the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, which acts in a dose‐ and time‐dependent manner, SCAMP1 (secretory carrier membrane protein 1) and FM4‐64 are mostly excluded from the SYP61‐positive BFA‐induced TGN aggregates, indicating that homotypic fusion of the TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE‐derived BFA‐induced aggregates. As the TGN also serves as an EE, continuously receiving materials from the plasma membrane, our data support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in terms of its response to BFA treatment in plant cells. Thus, the Golgi and TGN are probably functionally distinct organelles in plants.     

A Human Dynamin-related Protein Controls the Distribution of Mitochondria

Mitochondria exist as a dynamic tubular network with projections that move, break, and reseal in response to local environmental changes. We present evidence that a human dynamin-related protein (Drp1) is specifically required to establish this morphology. Drp1 is a GTPase with a domain structure similar to that of other dynamin family members. To identify the function of Drp1, we transiently transfected cells with mutant Drp1. A mutation in the GTPase domain caused profound alterations in mitochondrial morphology. The tubular projections normally present in wild-type cells were retracted into large perinuclear aggregates in cells expressing mutant Drp1. The morphology of other organelles was unaffected by mutant Drp1. There was also no effect of mutant Drp1 on the transport functions of the secretory and endocytic pathways. By EM, the mitochondrial aggregates found in cells that were transfected with mutant Drp1 appear as clusters of tubules rather than a large mass of coalescing membrane. We propose that Drp1 is important for distributing mitochondrial tubules throughout the cell. The function of this new dynamin-related protein in organelle morphology represents a novel role for a member of the dynamin family of proteins.     

Toxoplasma sortilin-like receptor regulates protein transport and is essential for apical secretory organelle biogenesis and host infection

Apicomplexan parasites have an assortment of unique apical secretory organelles (rhoptries and micronemes), which have crucial functions in host infection. Here, we show that a Toxoplasma gondii sortilin-like receptor (TgSORTLR) is required for the subcellular localization and formation of apical secretory organelles. TgSORTLR is a transmembrane protein that resides within Golgi-endosomal related compartments. The lumenal domain specifically interacts with rhoptry and microneme proteins, while the cytoplasmic tail of TgSORTLR recruits cytosolic sorting machinery involved in anterograde and retrograde protein transport. Ectopic expression of the N-terminal TgSORTLR lumenal domain results in dominant negative effects with the mislocalization of both endogenous TgSORTLR as well as rhoptry and microneme proteins. Conditional ablation of TgSORTLR disrupts rhoptry and microneme biogenesis, inhibits parasite motility, and blocks both invasion into and egress from host cells. Thus, the sortilin-like receptor is essential for protein trafficking and the biogenesis of key secretory organelles in Toxoplasma.     

Recycling of the dense-core vesicle membrane protein phogrin in Min6 beta-cells

Phogrin (IA2-beta) is an integral membrane protein of dense-core vesicles in neuroendocrine cells. We have examined the recycling of endogenous phogrin following exocytosis in insulin secreting Min6 beta-cells by monitoring stimulus dependent-uptake of antibodies directed against the lumenal domain of the protein. While low levels of internalized phogrin accumulated in LAMP1-positive lysosomes, more than 35% of internalized phogrin recycled back to an insulin-positive compartment and could return to the cell surface during a second exocytic stimulation. The recycling phogrin transited a syntaxin 6-positive compartment but did not appear to go through the TGN38-positive trans Golgi network. The results suggest a model in which secretory membrane components can recycle from the endosomal system to immature secretory granules without interaction with the major portion of the TGN.     

The relationship between lumenal and limiting membranes in swollen late endocytic compartments formed after wortmannin treatment or sucrose accumulation

Immunofluorescence and electron microscopy were used to evaluate the formation of swollen endosomes in NRK cells after treatment with wortmannin or sucrose and to study the relationship between lumenal and limiting membrane. Both treatments resulted in the formation of two populations of swollen late endocytic vacuoles, positive for lysosomal glycoproteins or cation-independent mannose 6-phosphate receptors, but those induced by wortmannin were characterised by time-dependent accumulation of lumenal vesicles, whereas those induced by sucrose uptake did not accumulate lumenal vesicles. In both cases, the distribution of the late endosomal marker, lysobisphosphatidic acid, remained unchanged and was present within the lumen of the swollen vacuoles. Consumption of plasma membrane and peripheral early endosomes, and the appearance of transferrin receptors in swollen late endosomes, indicated that continued membrane influx from early endocytic compartments, together with inhibition of membrane traffic out of the swollen compartments, is sufficient to account for the observed phenotype of cells treated with wortmannin. The accumulation of organelles with the characteristic morphology of endocytic carrier vesicles in cells that have taken up sucrose offers an explanation for the paucity of lumenal vesicles in swollen sucrosomes. Our data suggest that in fibroblast cells the swollen endosome phenotype induced by wortmannin is a consequence of endocytic membrane influx, coupled with the failure to recycle membrane to other cellular destinations, and not the inhibition of multivesicular body biogenesis.     

Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence

Protein localization to the TGN was investigated by examining the subcellular distribution of chimeric proteins in which the cytoplasmic and/or transmembrane domains of the TGN protein, TGN38, were substituted for the analogous domains of the plasma membrane protein, Tac. Using immunofluorescence and immunoelectron microscopy, the COOH- terminal cytoplasmic domain of TGN38 was found to be sufficient for localization of the chimeric proteins to the TGN. Deletion analysis identified an 11-amino acid segment containing the critical sequence, YQRL, as being sufficient for TGN localization. TGN localization was abrogated by mutation of the tyrosine or leucine residues in this sequence to alanine, or of the arginine residue to aspartate. In addition to specifying TGN localization, the 11-amino acid segment was active as an internalization signal, although the property of internalization alone was insufficient to confer TGN localization. Overexpression of chimeric proteins containing TGN localization determinants resulted in their detection at the plasma membrane and in intracellular vesicles, and abolished detection of endogenous TGN38. These results suggest that discrete cytoplasmic determinants can mediate protein localization to the TGN, and reveal a novel role for tyrosine-based motifs in this process.