Cytoplasmic phospholipase A2 antagonists inhibit multiple endocytic membrane trafficking pathways

Previous studies have suggested a role for cytosolic Ca²⁺-independent phospholipase A₂ (PLA₂) activity in the formation of endosome membrane tubules that participate in the export of transferrin (Tf) and transferrin receptors (TfR) from sorting endosomes (SEs) and the endocytic recycling compartment (ERC). Here we show that the PLA₂ requirement is a general feature of endocytic trafficking. The reversible cytoplasmic PLA₂ antagonist ONO-RS-082 (ONO) produced a concentration-dependent, differential block in the endocytic recycling of both low-density lipoprotein receptor (LDLR) and TfRs, and in the degradative pathways of LDL and epidermal growth factor (EGF). These results are consistent with the model that a cytoplasmic PLA₂ plays a general role in the export of cargo from multiple endocytic compartments by mediating the formation of membrane tubules.     

The phospholipase A₂ enzyme complex PAFAH Ib mediates endosomal membrane tubule formation and trafficking

Previous studies have shown that membrane tubule-mediated export from endosomal compartments requires a cytoplasmic phospholipase A(2) (PLA(2)) activity. Here we report that the cytoplasmic PLA(2) enzyme complex platelet-activating factor acetylhydrolase (PAFAH) Ib, which consists of α1, α2, and LIS1 subunits, regulates the distribution and function of endosomes. The catalytic subunits α1 and α2 are located on early-sorting endosomes and the central endocytic recycling compartment (ERC) and their overexpression, but not overexpression of their catalytically inactive counterparts, induced endosome membrane tubules. In addition, overexpression α1 and α2 altered normal endocytic trafficking; transferrin was recycled back to the plasma membrane directly from peripheral early-sorting endosomes instead of making an intermediate stop in the ERC. Consistent with these results, small interfering RNA-mediated knockdown of α1 and α2 significantly inhibited the formation of endosome membrane tubules and delayed the recycling of transferrin. In addition, the results agree with previous reports that PAFAH Ib α1 and α2 expression levels affect the distribution of endosomes within the cell through interactions with the dynein regulator LIS1. These studies show that PAFAH Ib regulates endocytic membrane trafficking through novel mechanisms involving both PLA(2) activity and LIS1-dependent dynein function.     

Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment

Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tf10) which was prepared by chemical cross- linking of transferrin (Tf). Tf10 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tf10 was investigated using fluorescence and electron microscopy. Tf10 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tf10 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf10. The presence of Tf10 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf10 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf10 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tf10 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.     

Rab4 Regulates Formation of Synaptic-like Microvesicles from Early Endosomes in PC12 Cells

Early endosomes in PC12 cells are an important site for the formation of synaptic-like microvesicles and constitutive recycling vesicles. By immunogold electron microscopy, the small GTPase rab4 was localized to early endosomes and numerous small vesicles in the cell periphery and Golgi area of PC12 cells. Overexpression of GTPase-deficient Q67Lrab4 increased the number of early endosome-associated and cytoplasmic vesicles, whereas expression of GDP-bound S22Nrab4 significantly increased the length of early endosomal tubules. In parallel, Q67Lrab4 induced a shift in rab4, VAMP2, and TfR label from early endosomes to peripheral vesicles, whereas S22Nrab4 increased early endosome labeling of all three proteins. These observations were corroborated by early endosome budding assays. Together, our data document a thus far unrecognized role for rab4 in the formation of synaptic-like microvesicles and add to our understanding of the formation of constitutive recycling vesicles from early endosomes.     

Rab22a regulates the recycling of membrane proteins internalized independently of clathrin

Plasma membrane proteins that are internalized independently of clathrin, such as major histocompatibility complex class I (MHCI), are internalized in vesicles that fuse with the early endosomes containing clathrin-derived cargo. From there, MHCI is either transported to the late endosome for degradation or is recycled back to the plasma membrane via tubular structures that lack clathrin-dependent recycling cargo, e.g., transferrin. Here, we show that the small GTPase Rab22a is associated with these tubular recycling intermediates containing MHCI. Expression of a dominant negative mutant of Rab22a or small interfering RNA-mediated depletion of Rab22a inhibited both formation of the recycling tubules and MHCI recycling. By contrast, cells expressing the constitutively active mutant of Rab22a exhibited prominent recycling tubules and accumulated vesicles at the periphery, but MHCI recycling was still blocked. These results suggest that Rab22a activation is required for tubule formation and Rab22a inactivation for final fusion of recycling membranes with the surface. The trafficking of transferrin was only modestly affected by these treatments. Dominant negative mutant of Rab11a also inhibited recycling of MHCI but not the formation of recycling tubules, suggesting that Rab22a and Rab11a might coordinate different steps of MHCI recycling.     

Transferrin receptor 2: evidence for ligand-induced stabilization and redirection to a recycling pathway

Transferrin receptor 2 (TfR2) is a homologue of transferrin receptor 1 (TfR1), the protein that delivers iron to cells through receptor-mediated endocytosis of diferric transferrin (Fe(2)Tf). TfR2 also binds Fe(2)Tf, but it seems to function primarily in the regulation of systemic iron homeostasis. In contrast to TfR1, the trafficking of TfR2 within the cell has not been extensively characterized. Previously, we showed that Fe(2)Tf increases TfR2 stability, suggesting that trafficking of TfR2 may be regulated by interaction with its ligand. In the present study, therefore, we sought to identify the mode of TfR2 degradation, to characterize TfR2 trafficking, and to determine how Fe(2)Tf stabilizes TfR2. Stabilization of TfR2 by bafilomycin implies that TfR2 traffics to the lysosome for degradation. Confocal microscopy reveals that treatment of cells with Fe(2)Tf increases the fraction of TfR2 localizing to recycling endosomes and decreases the fraction of TfR2 localizing to late endosomes. Mutational analysis of TfR2 shows that the mutation G679A, which blocks TfR2 binding to Fe(2)Tf, increases the rate of receptor turnover and prevents stabilization by Fe(2)Tf, indicating a direct role of Fe(2)Tf in TfR2 stabilization. The mutation Y23A in the cytoplasmic domain of TfR2 inhibits its internalization and degradation, implicating YQRV as an endocytic motif.     

Receptor-mediated endocytosis and intracellular trafficking of lipoproteins and transferrin in insect cells

While the intracellular pathways of ligands after receptor-mediated endocytosis have been studied extensively in mammalian cells, in insect cells these pathways are largely unknown. We transfected Drosophila Schneider line 2 (S2) cells with the human low-density lipoprotein (LDL) receptor (LDLR) and transferrin (Tf) receptor (TfR), and used endocytosis of LDL and Tf as markers. After endocytosis in mammalian cells, LDL is degraded in lysosomes, whereas Tf is recycled. Fluorescence microscopy analysis revealed that LDL and Tf are internalized by S2 cells transfected with LDLR or TfR, respectively. In transfectants simultaneously expressing LDLR and TfR, both ligands colocalize in endosomes immediately after endocytic uptake, and their location remained unchanged after a chase. Similar results were obtained with Spodoptera frugiperda Sf9 cells that were transfected with TfR, suggesting that Tf is retained intracellularly by both cell lines. The insect lipoprotein, lipophorin, is recycled upon lipophorin receptor (LpR)-mediated endocytosis by mammalian cells, however, not after endocytosis by LpR-expressing S2 transfectants, suggesting that this recycling mechanism is cell-type specific. LpR is endogenously expressed by fat body tissue of Locusta migratoria for a limited period after an ecdysis. A chase following endocytosis of labeled lipophorin by isolated fat body tissue at this developmental stage resulted in a significant decrease of lipophorin-containing vesicles, indicative of recycling of the ligand.     

Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump

Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosomes acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cation-dependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluid-phase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Baf), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-containing endocytic compartments in living cells revealed elevated endosomal pH values (pH > 7.0) within 2 min after addition of Baf. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +/- 0.08 and 1.57 +/- 0.25, respectively. Consequently, little if any in TfR expression at the cell surface was measured during Baf treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'- diaminobenzidine cross-linking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Baf, sorting was initiated after 20 min of uptake, reaching approximately 40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nearly completely abrogated transport to cathepsin D- labeled lysosomes. From these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the vacuolar proton pump.     

A role for calmodulin in organelle membrane tubulation.

Membrane tubules of uniform diameter (60-80 nm) and variable lengths have been seen to extend from the main bodies of the Golgi complex, trans Golgi network (TGN), and endosomes. In the case of endosomes, these tubules appear to mediate membrane and receptor recycling events. Brefeldin A (BFA) is a potent drug that completely blocks coated vesicle formation from the Golgi complex and TGN, but at the same time causes the enhanced formation of membrane tubules from these same organelles. Recently, experiments have shown that calmodulin antagonists inhibit the transport of receptors out of endosomes, perhaps by inhibiting the formation of recycling tubules. Using the potent calmodulin-specific antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13), and N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide (C-1), we found that the recycling of transferrin from endosomes to the cell surface was significantly inhibited, resulting in the formation of enlarged endosomal vacuoles. In addition, these same calmodulin antagonists also potently inhibited the formation of BFA-stimulated membrane tubules from the Golgi complex, TGN, and endosomes. In the case of the Golgi complex, failure to form tubules resulted in the inhibition of BFA-stimulated retrograde transport to the endoplasmic reticulum. These results suggest that calmodulin is a general regulator of membrane tubulation and is capable of influencing the morphology of several organelles.     

Fusion accessibility of endocytic compartments along the recycling and lysosomal endocytic pathways in intact cells

A fluorescence assay developed for the quantitation of intracellular fusion of sequentially formed endocytic compartments (Salzman, N. H., and F. R. Maxfield. 1988 J. Cell Biol. 106:1083-1091) has been used to measure the time course of endosome fusion accessibility along the recycling and degradative endocytic pathways. Transferrin (Tf) was used to label the recycling pathway, and alpha2-macroglobulin (alpha 2 M) was used to label the lysosomal degradative pathway. Along the degradative pathway, accessibility of vesicles containing alpha 2M to fusion with subsequently formed endocytic vesicles decreased with apparent first order kinetics. The t12 for the loss of fusion accessibility was approximately 8 min. The behavior of Tf is more complex. Initially the fusion accessibility of Tf decayed rapidly (t1/2 less than 3 min), but a constant level of fusion accessibility was then observed for 10 min. This suggests that Tf moves through one fusion accessible endosome rapidly and then enters a second fusion accessible compartment on the recycling pathway. At 18 degrees C, fusion of antifluorescein antibodies (AFA) containing vesicles with F-alpha 2M was observed when the interval between additions was 10 min. However, if the interval was increased to 1 h, no fusion with incoming vesicles was observed. These results identify the site of F-alpha 2M accumulation at 18 degrees C as a prelysosomal late endosome that no longer fuses with newly formed endosomes since no delivery to lysosomes is observed at this temperature.     

The GTP/GDP cycling of rho GTPase TCL is an essential regulator of the early endocytic pathway

Rho GTPases are key regulators of actin dynamics. We report that the Rho GTPase TCL, which is closely related to Cdc42 and TC10, localizes to the plasma membrane and the early/sorting endosomes in HeLa cells, suggesting a role in the early endocytic pathway. Receptor-dependent internalization of transferrin (Tf) is unaffected by suppression of endogenous TCL by small interfering RNA treatment. However, Tf accumulates in Rab5-positive uncoated endocytic vesicles and fails to reach the early endosome antigen-1-positive early endosomal compartments and the pericentriolar recycling endosomes. Moreover, Tf release upon TCL knockdown is significantly slower. Conversely, in the presence of dominant active TCL, internalized Tf accumulates in early endosome antigen-1-positive early/sorting endosomes and not in perinuclear recycling endosomes. Tf recycles directly from the early/sorting endosomes and it is normally released by the cells. The same phenotype is generated by replacing the C terminus of dominant active Cdc42 and TC10 with that of TCL, indicating that all three proteins share downstream effector proteins. Thus, TCL is essential for clathrin-dependent endocytosed receptors to enter the early/sorting endosomes. Furthermore, the active GTPase favors direct recycling from early/sorting endosomes without accumulating in the perinuclear recycling endosomes.     

Sphingomyelin synthase 1-generated sphingomyelin plays an important role in transferrin trafficking and cell proliferation

Transferrin (Tf) endocytosis and recycling are essential for iron uptake and the regulation of cell proliferation. Tf and Tf receptor (TfR) complexes are internalized via clathrin-coated pits composed of a variety of proteins and lipids and pass through early endosomes to recycling endosomes. We investigated the role of sphingomyelin (SM) synthases (SMS1 and SMS2) in clathrin-dependent trafficking of Tf and cell proliferation. We employed SM-deficient lymphoma cells that lacked SMSs and that failed to proliferate in response to Tf. Transfection of SMS1, but not SMS2, enabled these cells to incorporate SM into the plasma membrane, restoring Tf-mediated proliferation. SM-deficient cells showed a significant reduction in clathrin-dependent Tf uptake compared with the parental SM-producing cells. Both SMS1 gene transfection and exogenous short-chain SM treatment increased clathrin-dependent Tf uptake in SM-deficient cells, with the Tf being subsequently sorted to Rab11-positive recycling endosomes. We observed trafficking of the internalized Tf to late/endolysosomal compartments, and this was not dependent on the clathrin pathway in SM-deficient cells. Thus, SMS1-mediated SM synthesis directs Tf-TfR to undergo clathrin-dependent endocytosis and recycling, promoting the proliferation of lymphoma cells.     

Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20 degrees C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.     

Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome

To study the fusion and separation of endocytic compartments, we have used digital image analysis to quantify the accumulation of fluorescent ligands in endosomes during continuous endocytosis for periods of 1-20 min. Fluorescently labeled transferrin (Tf) and low density lipoproteins (LDL) were used as markers of recycling receptors and lysosomally directed ligands respectively. By measuring the intensity of individual endosomes, we found that the amount of LDL per endosome increases 30-40-fold between 1 and 10 min and then plateaus. In contrast, the amount of Tf per endosome reaches a steady state within 2 min at a level that is only three to four times that at 1 min. We used pulse-chase double label methods to demonstrate that Tf cycles through the compartment in which the LDL accumulates. When both Tf and LDL are added to cells simultaneously for 2 min, nearly all endosomes contain both labels. With 2-4 min further incubation in the absence of external ligands, LDL-containing compartments become depleted of Tf as Tf is directed to para-Golgi recycling endosomes. However, if Tf is added to the medium 2-4 min after a pulse with LDL, most of the LDL-containing endosomes become labeled with Tf. The data indicate that at least 30-40 endocytic vesicles containing both Tf and LDL fuse with an endosomal compartment over a period of 5-10 min. LDL accumulates within this compartment and Tf is simultaneously removed. Simple mathematical models suggest that this type of iterative fractionation can lead to very high efficiency sorting.     

Transferrin receptor 2 mediates a biphasic pattern of transferrin uptake associated with ligand delivery to multivesicular bodies

The physiological role of transferrin (Tf) receptor 2 (TfR2), a homolog of the well-characterized TfR1, is unclear. Mutations in TfR2 result in hemochromatosis, indicating that this receptor has a unique role in iron metabolism. We report that HepG2 cells, which endogenously express TfR2, display a biphasic pattern of Tf uptake when presented with ligand concentrations up to 2 µM. The apparently nonsaturating pathway of Tf endocytosis resembles TfR1-independent Tf uptake, a process previously characterized in some liver cell types. Exogenous expression of TfR2 but not TfR1 induces a similar biphasic pattern of Tf uptake in HeLa cells, supporting a role for TfR2 in this process. Immunoelectron microscopy reveals that while Tf, TfR1, and TfR2 are localized in the plasma membrane and tubulovesicular endosomes, TfR2 expression is associated with the additional appearance of Tf in multivesicular bodies. These combined results imply that unlike TfR1, which recycles apo-Tf back to the cell surface after the release of iron, TfR2 promotes the intracellular deposition of ligand. Tf delivered by TfR2 does not appear to be degraded, which suggests that its delivery to this organelle may be functionally relevant to the storage of iron in overloaded states. iron transport; HepG2 cells     

Inhibition of a Golgi complex lysophospholipid acyltransferase induces membrane tubule formation and retrograde trafficking

Recent studies have suggested that formation of Golgi membrane tubules involves the generation of membrane-associated lysophospholipids by a cytoplasmic Ca2+-independent phospholipase A2 (PLA2). Herein, we provide additional support for this idea by showing that inhibition of lysophospholipid reacylation by a novel Golgi-associated lysophosphatidylcholine acyltransferase (LPAT) induces the rapid tubulation of Golgi membranes, leading in their retrograde movement to the endoplasmic reticulum. Inhibition of the Golgi LPAT was achieved by 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976), a previously characterized antagonist of acyl-CoA cholesterol acyltransferase. The effect of CI-976 was similar to that of brefeldin A, except that the coatomer subunit beta-COP remained on Golgi-derived membrane tubules. CI-976 also enhanced the cytosol-dependent formation of tubules from Golgi complexes in vitro and increased the levels of lysophosphatidylcholine in Golgi membranes. Moreover, preincubation of cells with PLA2 antagonists inhibited the ability of CI-976 to induce tubules. These results suggest that Golgi membrane tubule formation can result from increasing the content of lysophospholipids in membranes, either by stimulation of a PLA2 or by inhibition of an LPAT. These two opposing enzyme activities may help to coordinately regulate Golgi membrane shape and tubule formation.     

Inhibition of membrane tubule formation and trafficking by isotetrandrine, an antagonist of G-protein-regulated phospholipase A2 enzymes

Previous studies have established a role for cytoplasmic phospholipase A(2) (PLA(2)) activity in tubule-mediated retrograde trafficking between the Golgi complex and the endoplasmic reticulum (ER). However, little else is known about how membrane tubule formation is regulated. This study demonstrates that isotetrandrine (ITD), a biscoclaurine alkaloid known to inhibit PLA(2) enzyme activation by heterotrimeric G-proteins, effectively prevented brefeldin A (BFA)-induced tubule formation from the Golgi complex and retrograde trafficking to the ER. In addition, ITD inhibited BFA-stimulated tubule formation from the trans-Golgi network and endosomes. ITD inhibition of the BFA response was potent (IC(50) approximately 10-20 microM) and rapid (complete inhibition with a 10-15-min preincubation). ITD also inhibited normal retrograde trafficking as revealed by the formation of nocodazole-induced Golgi mini-stacks at ER exit sites. Treatment of cells with ITD alone caused the normally interconnected Golgi ribbons to become fragmented and dilated, but cisternae were still stacked and located in a juxtanuclear position. These results suggest that a G-protein-binding PLA(2) enzyme plays a pivotal role in tubule mediated trafficking between the Golgi and the ER, the maintenance of the interconnected ribbons of Golgi stacks, and tubule formation from endosomes.     

Gyrating clathrin: highly dynamic clathrin structures involved in rapid receptor recycling

We report here detection of novel intracellular clathrin-coated structures revealed by continuous high-speed imaging of cells expressing green fluorescent protein fusion proteins. These structures, which we operationally term 'gyrating clathrin' (G-clathrin), are characterized by localized but extremely rapid movement, leading to the hypothesis that they are coated buds on waving membrane tubules. G-clathrin structures have structurally and functionally distinct features. They lack detectable adaptor proteins AP-1 and AP-2 but contain GGA1 [Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding protein] as well as the cation-dependent mannose-6-phosphate receptor. While they accumulate internalized transferrin (Tf), they do not contain detectable levels of cargos targeted for the late endosome/lysosome pathway such as EGF and dextran. Pulse-chase studies indicate that Tf appears in G-clathrin structures in the cell periphery after sorting endosomes (SEs), but before filling of the perinuclear endocytic recycling compartment. Furthermore, the inhibitors LY294002 and wortmannin, which inhibit direct recycling of Tf from SEs to the plasma membrane, also block its appearance in G-clathrin. These observations suggest that peripheral G-clathrin contributes to rapid recycling, a kinetically defined compartment that has largely eluded structural identification. More generally, the rapid continuous live cell imaging reported here reveals new aspects of membrane trafficking.     

SNX-BAR-mediated endosome tubulation is co-ordinated with endosome maturation

Endosomal sorting is essential for cell homeostasis. Proteins targeted for degradation are retained in the maturing endosome vacuole while others are recycled to the cell surface or sorted to the biosynthetic pathway via tubular transport carriers. Sorting nexin (SNX) proteins containing a BAR (for Bin-Amphiphysin-Rvs) domain are key regulators of phosphoinositide-mediated, tubular-based endosomal sorting, but how such sorting is co-ordinated with endosomal maturation is not known. Here, using well-defined Rab GTPases as endosomal compartment markers, we have analyzed the localization of SNX1 [endosome-to-trans-Golgi network (TGN) transport as part of the SNX-BAR-retromer complex], SNX4 (cargo-recycling from endosomes to the plasma membrane) and SNX8 (endosomes-to-TGN trafficking in a retromer-independent manner). We show that these SNX-BARs are primarily localized to early endosomes, but display the highest frequency of tubule formation at the moment of early-to-late endosome transition: the Rab5-to-Rab7 switch. Perturbing this switch shifts SNX-BAR tubulation to early endosomes, resulting in SNX1-decorated tubules that lack retromer components VPS26 and VPS35, suggesting that both early and late endosomal characteristics of the endosome are important for SNX-BAR-retromer-tubule formation. We also establish that SNX4, but not SNX1 and SNX8, is associated with the Rab11-recycling endosomes and that a high frequency of SNX4-mediated tubule formation is observed as endosomes undergo Rab4-to-Rab11 transition. Our study therefore provides evidence for fine-tuning between the processes of endosomal maturation and the formation of endosomal tubules. As tubulation is required for SNX1-, SNX4- and SNX8-mediated sorting, these data reveal a previously unrecognized co-ordination between maturation and tubular-based sorting.