A novel TIP30 protein complex regulates EGF receptor signaling and endocytic degradation

Activated epidermal growth factor receptor (EGFR) continues to signal in the early endosome, but how this signaling process is regulated is less well understood. Here we describe a protein complex consisting of TIP30, endophilin B1, and acyl-CoA synthetase long chain family member 4 (ACSL4) that interacts with Rab5a and regulates EGFR endocytosis and signaling. These proteins are required for the proper endocytic trafficking of EGF-EGFR. Knockdown of TIP30, ACSL4, endophilin B1, or Rab5a in human liver cancer cells or genetic knock-out of Tip30 in mouse primary hepatocytes results in the trapping of EGF-EGFR complexes in early endosomes, leading to delayed EGFR degradation and prolonged EGFR signaling. Furthermore, we show that Rab5a colocalizes with vacuolar (H(+))-ATPases (V-ATPases) on transport vesicles. The TIP30 complex facilitates trafficking of Rab5a and V-ATPases to EEA1-positive endosomes in response to EGF. Together, these results suggest that this TIP30 complex regulates EGFR endocytosis by facilitating the transport of V-ATPases from trans-Golgi network to early endosomes.     

SPIN90 Knockdown Attenuates the Formation and Movement of Endosomal Vesicles in the Early Stages of Epidermal Growth Factor Receptor Endocytosis

The finding that SPIN90 colocalizes with epidermal growth factor (EGF) in EEA1-positive endosomes prompted us to investigate the role of SPIN90 in endocytosis of the EGF receptor (EGFR). In the present study, we demonstrated that SPIN90 participates in the early stages of endocytosis, including vesicle formation and trafficking. Stable HeLa cells with knockdown of SPIN90 displayed significantly higher levels of surface EGFR than control cells. Analysis of the abundance and cellular distribution of EGFR via electron microscopy revealed that SPIN90 knockdown cells contain residual EGFR at cell membranes and fewer EGFR-containing endosomes, both features that reflect reduced endosome formation. The delayed early endosomal targeting capacity of SPIN90 knockdown cells led to increased EGFR stability, consistent with the observed accumulation of EGFR at the membrane. Small endosome sizes and reduced endosome formation in SPIN90 knockdown cells, observed using fluorescent confocal microscopy, strongly supported the involvement of SPIN90 in endocytosis of EGFR. Overexpression of SPIN90 variants, particularly the SH3, PRD, and CC (positions 643 - 722) domains, resulted in aberrant morphology of Rab5-positive endosomes (detected as small spots located near the cell membrane) and defects in endosomal movement. These findings clearly suggest that SPIN90 participates in the formation and movement of endosomes. Consistent with this, SPIN90 knockdown enhanced cell proliferation. The delay in EGFR endocytosis effectively increased the levels of endosomal EGFR, which triggered activation of ERK1/2 and cell proliferation via upregulation of cyclin D1. Collectively, our findings suggest that SPIN90 contributes to the formation and movement of endosomal vesicles, and modulates the stability of EGFR protein, which affects cell cycle progression via regulation of the activities of downstream proteins, such as ERK1/2, after EGF stimulation.     

Re-localization of activated EGF receptor and its signal transducers to multivesicular compartments downstream of early endosomes in response to EGF

The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction. However, accumulating data indicate that signal transduction from internalized cell surface receptors also occurs from endosomes. We show that a substantial fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGFR) and Shc, Grb2 and Cbl after internalization relocates from early endosomes to compartments which are negative for the early endosomes, recycling vesicle markers EEA1 and transferrin in EGF-stimulated cells. These compartments contained the multivesicular body and late endosome marker CD63, and the late endosome and lysosome marker LAMP-1, and showed a multivesicular morphology. Subcellular fractionation revealed that activated EGFR, adaptor proteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-positive fractions. Co-immunoprecipitations showed EGFR in complex with both Shc, Grb2 and Cbl. Treatment with the weak base chloroquine or inhibitors of lysosomal enzymes after EGF stimulation induced an accumulation of tyrosine-phosphorylated EGFR and Shc in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activation of ERK 1 and 2. These results suggest that EGFR signaling is not spatially restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.     

Engagement of the Small GTPase Rab31 Protein and Its Effector,Early Endosome Antigen 1, Is Important for Trafficking of the Ligand-bound Epidermal Growth Factor Receptor from the Early to the Late Endosome

Rab31 is a member of the Rab5 subfamily of Rab GTPases. Although localized largely to the trans-Golgi network, it shares common guanine nucleotide exchange factors and effectors with other Rab5 subfamily members that have been implicated in endocytic membrane traffic. We investigated whether Rab31 also has a role in the trafficking of the ligand-bound EGF receptor (EGFR) internalized through receptor-mediated endocytosis. We found that loss of Rab31 inhibits, but overexpression enhances, EGFR trafficking to the late endosomes and that the effect of Rab31 silencing could be specifically rescued by overexpression of a silencing-resistant form of Rab31. Rab31 was found to interact with the EGFR by coimmunoprecipitation and affinity pulldown analyses, and the primarily trans-Golgi network-localized Rab31 has increased colocalization with the EGFR in A431 cells 30 min after pulsing with EGF. A glycerol gradient sedimentation assay suggested that Rab31 is sequestered into a high molecular weight complex after stimulation with EGF, as was early endosome antigen 1 (EEA1), a factor responsible for endosomal tethering and fusion events. We found that loss of EEA1 reduced the interaction between Rab31 and the EGFR and abrogated the effect of Rab31 overexpression on the trafficking of the EGFR. Likewise, loss of GAPex5, a Rab31 guanine nucleotide exchange factor that has a role in ubiquitination and degradation of the EGFR, reduced the interaction of Rab31 with the EGFR and its effect on EGFR trafficking. Taken together, our results suggest that Rab31 is an important regulator of endocytic trafficking of the EGFR and functions in an EGFR trafficking complex that includes EEA1 and GAPex5.     

CD2AP/CMS regulates endosome morphology and traffic to the degradative pathway through its interaction with Rab4 and c-Cbl

The small GTPase Rab4 is involved in endocytosis through sorting and recycling early endosomes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for effectors that specifically interact with Rab4-Q67L, the GTP-bound form of Rab4. We cloned an ubiquitous 80-kDa protein, identical to CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS), that interacts with Rab4-Q67L in the yeast two-hybrid system and in vitro . CD2AP/CMS expressed in mammalian cells was localized to punctate structures and along actin filaments. None of the known markers of early endosomes [Early Endosomes Antigen 1 (EEA1), Rab5 and Rab11] colocalized with the CD2AP/CMS-positive vesicles. However, coexpression of Rab4-Q67L with CD2AP/CMS induces a significant enlargement of EEA1-positive early endosomes. Rab4, CD2AP/CMS and Rab7 colocalized in these modified endosomes. Coexpression of c-Cbl and CD2AP/CMS also resulted in an enlargement of early endosomes. Using various truncated forms of CD2AP/CMS, we demonstrate that early endosomes enlargement requires that CD2AP/CMS interacts with both Rab4 and c-Cbl. The expression of a truncated form of CD2AP/CMS that retains the ability to interact with Rab4 but not c-Cbl inhibits ligand-induced PDGF receptor degradation. We propose that CD2AP/CMS, through interactions with Rab4 and c-Cbl, controls early endosome morphology and may play a role in traffic between early and late endosomes, and thus in the degradative pathway .     

The Arf6 GTPase-activating Proteins ARAP2 and ACAP1 Define Distinct Endosomal Compartments That Regulate Integrin α5β1 Traffic

Arf6 and the Arf6 GTPase-activating protein (GAP) ACAP1 are established regulators of integrin traffic important to cell adhesion and migration. However, the function of Arf6 with ACAP1 cannot explain the range of Arf6 effects on integrin-based structures. We propose that Arf6 has different functions determined, in part, by the associated Arf GAP. We tested this idea by comparing the Arf6 GAPs ARAP2 and ACAP1. We found that ARAP2 and ACAP1 had opposing effects on apparent integrin β1 internalization. ARAP2 knockdown slowed, whereas ACAP1 knockdown accelerated, integrin β1 internalization. Integrin β1 association with adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif (APPL)-positive endosomes and EEA1-positive endosomes was affected by ARAP2 knockdown and depended on ARAP2 GAP activity. ARAP2 formed a complex with APPL1 and colocalized with Arf6 and APPL in a compartment distinct from the Arf6/ACAP1 tubular recycling endosome. In addition, although ACAP1 and ARAP2 each colocalized with Arf6, they did not colocalize with each other and had opposing effects on focal adhesions (FAs). ARAP2 overexpression promoted large FAs, but ACAP1 overexpression reduced FAs. Taken together, the data support a model in which Arf6 has at least two sites of opposing action defined by distinct Arf6 GAPs.     

Epidermal growth factor and membrane trafficking. EGF receptor activation of endocytosis requires Rab5a

Activated epidermal growth factor receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between signaling and endocytosis is not well understood. Here we show that EGF stimulation of NR6 cells induces a specific, rapid and transient activation of Rab5a. EGF also enhanced translocation of the Rab5 effector, early endosomal autoantigen 1 (EEA1), from cytosol to membrane. The activation of endocytosis, fluid phase and receptor mediated, by EGF was enhanced by Rab5a expression, but not by Rab5b, Rab5c, or Rab5a truncated at the NH(2) and/or COOH terminus. Dominant negative Rab5a (Rab5:N34) blocked EGF-stimulated receptor-mediated and fluid-phase endocytosis. EGF activation of Rab5a function was dependent on tyrosine residues in the COOH-terminal domain of the EGF receptor (EGFR). Removal of the entire COOH terminus by truncation (c'973 and c'991) abrogated ligand-induced Rab5a activation of endocytosis. A "kinase-dead" EGFR failed to stimulate Rab5a function. However, another EGF receptor mutant (c'1000), with the kinase domain intact and a single autophosphorylation site effectively signaled Rab5 activation. These results indicate that EGFR and Rab5a are linked via a cascade that results in the activation of Rab5a and that appears essential for internalization. The results point to an interdependent relationship between receptor activation, signal generation and endocytosis.     

Direct interaction of EEA1 with Rab5b.

The early endosomal autoantigen EEA1 is essential for early endosomal membrane fusion. It binds to endosomes via a C-terminal domain (EEA1-CT). To identify proteins interacting with EEA1-CT, we screened a human brain library in the yeast two-hybrid system. Fourteen clones reacted strongly with EEA1-CT. Sequencing of these clones revealed that they all contained the ORF of the small GTPase, Rab5b. Further two-hybrid analysis suggested that Rab5b also interacts with the N-terminus of EEA1 (EEA1-NT). The interaction of both EEA1-CT and EEA1-NT with Rab5b was confirmed biochemically, and was found to be GTP dependent. Confocal immunofluorescence microscopy indicated that EEA1 colocalizes with Rab5b on early endosomes. Although EEA1-CT and EEA1-NT interacted strongly with wild-type Rab5b in the two-hybrid system, we detected no interaction with wild-type Rab5a, even though GTPase-deficient mutants of both Rab5a and Rab5b interacted equally well with EEA1. This difference could not be explained by differences in intrinsic GTPase activities, as these were found to be very similar. Instead, we speculate that yeast may contain a GTPase-activating protein (GAP) activity that stimulates Rab5a but not Rab5b. In contrast, pig brain cytosol was found to contain a GAP activity that stimulates the GTPase activity of Rab5b in preference to that of Rab5a. These data provide evidence that EEA1 interacts with both Rab5a and Rab5b, and that the GTPase activities of the two proteins are differentially regulated in vivo.     

Regulation of epidermal growth factor receptor endocytosis by wortmannin through activation of Rab5 rather than inhibition of phosphatidylinositol 3-kinase

The involvement of phosphatidylinositol 3-kinase (PI3K) in membrane trafficking in mammalian cells has largely come from experiments with wortmannin. This compound inhibits endosome fusion in vitro, possibly by inhibiting the production of phosphatidylinositol (PtdIns)-3-P, which co-regulates EEA1 with Rab5. However, the results from wortmannin inhibition experiments performed in vivo differ significantly. We have recently shown that wortmannin enlarges endosomes containing the epidermal growth factor receptor (EGFR) and enhances the lysosomal degradation of EGFR. In this report, we demonstrate that addition of the PI3K reaction products does not suppress wortmannin-induced enlargement of EGFR-containing endosomes and enhancement of EGFR degradation. Moreover, the effects of wortmannin on the intracellular trafficking of EGFR mimic those of the permanently activated Rab5 mutant, Rab5 Q79L, which stimulates endosome fusion. We also found that an inactive Rab5 mutant, Rab5 S34N, blocks wortmannin-induced endosome enlargement and that wortmannin stimulates the activation of Rab5. We further showed that wortmannin reduced the membrane association of p120 Ras GTPase-activating protein (GAP) and inhibited the interaction between Rab5 and p120 Ras GAP. We conclude that wortmannin alters intracellular trafficking of EGFR by activating Rab5 rather than by inhibiting PI3K.     

Modulation of host cell endocytosis by the type III cytotoxin, Pseudomonas ExoS

Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin that possesses Rho GTPase-activating protein (RhoGAP) and ADP-ribosyltransferase (ADPr) activities. In the current study, the RhoGAP and ADPr activities of ExoS were tested for the ability to disrupt mammalian epithelial cell physiology. RhoGAP, but not ADPr, inhibited internalization/phagocytosis of bacteria, while ADPr, but not RhoGAP, inhibited vesicle trafficking, both general fluid-phase uptake and EGF-activated EGF receptor (EGFR) degradation. In ADPr-intoxicated cells, upon EGF activation, EGFR co-localized with clathrin-coated vesicles (CCV), which did not mature into Rab5-positive early endosomes. Constitutively, active Rab5 recruited EGFR from CCV to early endosomes. Consistent with the inhibition of Rab5 function by ADPr, several Rab proteins including Rab5 and 9, but not Rab4, were ADP ribosylated by ExoS. Thus, the two enzymatic activities of ExoS have different effects on epithelial cells with RhoGAP inhibiting bacterial internalization and ADPr interfering with CCV maturation. The ability ADPr to inhibit mammalian vesicle trafficking provides a new mechanism for bacterial toxin-mediated virulence.     

Biochemical characterization of APPL endosomes: the role of annexin A2 in APPL membrane recruitment

APPL endosomes are a recently identified subpopulation of early endosomes characterized by the presence of two homologous Rab5 effector proteins APPL1 and APPL2. They exhibit only limited colocalization with EEA1, another Rab5 effector and a marker of the canonical early endosomes. Although APPL endosomes appear to play important roles in cargo trafficking and signal transduction, their protein composition and biochemical properties remain largely unknown. Here we employed membrane fractionation methods to characterize APPL endosomes biochemically. We demonstrate that they represent heterogeneous membrane structures which can be discriminated from the canonical EEA1-positive early endosomes by their partly different physical properties and a distinct migration pattern in the continuous density gradients. In search for other potential markers of APPL endosomes we identified Annexin A2 as an interacting partner of both APPL1 and APPL2. Annexin A2 is a Ca(2+) and phosphatidylinositol 4,5-bisphosphate binding protein, previously implicated in several endocytic steps. We show that Annexin A2 co-fractionates and colocalizes with APPL endosomes. Moreover, silencing of its expression causes solubilization of APPL2 from endosomes. Although Annexin A2 is not an exclusive marker of APPL endosomes, our data suggest that it has an important function in membrane recruitment of APPL proteins, acting in parallel to Rab5.     

A GTPase-activating protein controls Rab5 function in endocytic trafficking

Rab-family GTPases are conserved regulators of membrane trafficking that cycle between inactive GDP-bound and activated GTP-bound states. A key determinant of Rab function is the lifetime of the GTP-bound state. As Rabs have a low intrinsic rate of GTP hydrolysis, this process is under the control of GTP-hydrolysis-activating proteins (GAPs). Due to the large number of Rabs and GAPs that are encoded by the human genome, it has proven difficult to assign specific functional relationships to these proteins. Here, we identify a Rab5-specific GAP (RabGAP-5), and show that RN-Tre (previously described as a Rab5 GAP) acts on Rab41. RabGAP-5 overexpression triggers a loss of the Rab5 effector EEA1 from endosomes and blocks endocytic trafficking. By contrast, depletion of RabGAP-5 results in increased endosome size, more endosome-associated EEA1, and disrupts the trafficking of EGF and LAMP1. RabGAP-5 therefore limits the amount of activated Rab5, and thereby regulates trafficking through endosomes.     

GAPex-5 mediates ubiquitination, trafficking, and degradation of epidermal growth factor receptor

Upon ligand stimulation, epidermal growth factor receptor (EGFR) is rapidly ubiquitinated, internalized, and sorted to lysosomes for degradation. Rab5 has been shown to play an important role in the early stages of EGFR trafficking. GAPex-5 is a newly described Rab5 exchange factor. Herein, we investigate the role of GAPex-5 on EGFR trafficking and degradation. Down-regulation of GAPex-5 by RNA interference decreases epidermal growth factor-stimulated EGFR degradation. Moreover, ubiquitination of EGFR is impaired by depletion of GAPex-5. This inhibitory effect is due to a decrease in the interaction between the adapter protein c-Cbl and EGFR, but not the phosphorylation state of EGFR. Consistently, when examined by immunofluorescence microscopy in cells depleted of GAPex-5, ligand-bound EGFR appeared trapped in early endosomes and the trafficking of internalized receptor from early to late endosomes was impaired. In agreement with the depletion studies, EGFR degradation is enhanced by overexpressing GAPex-5 wild type, but not GAPex-5DeltaGAP, a mutant lacking the Ras GTPase-activating protein (GAP) domain. This is consistent with the finding that c-Cbl binds specifically to the Ras GAP domain. Finally, overexpression of dominant negative Rab5a or depletion of all three isoforms of Rab5 does not inhibit ubiquitination of EGFR, which suggests that GAPex-5-mediated EGFR ubiquitination is independent of Rab5 activation. Collectively, the results suggest a novel mechanism by which EGF-stimulated receptor ubiquitination and trafficking are mediated via GAPex-5.     

Regulation of intracellular trafficking of the EGF receptor by Rab5 in the absence of phosphatidylinositol 3-kinase activity

Rab5 and phosphatidylinositol 3-kinase (PI3K) have been proposed to co-regulate receptor endocytosis by controlling early endosome fusion. However, in this report we demonstrate that inhibition of epidermal growth factor (EGF)-stimulated PI3K activity by expression of the kinase-deficient PI3K p110 subunit (p110Δkin) does not block the lysosomal targeting and degradation of the EGF receptor (EGFR). Moreover, inhibition of total PI3K activity by wortmannin or LY294002 significantly enlarges EGFR-containing endosomes and dissociates the early-endosomal autoantigen EEA1 from membrane fractions. However, this does not block the lysosomal targeting and degradation of EGFR. In contrast, transfection of cells with mutant Rab5 S34N or microinjection of anti-Rabaptin5 antibodies inhibits EGFR endocytosis. Our results, therefore, demonstrate that PI3K is not universally required for the regulation of receptor intracellular trafficking. The present work suggests that the intracellular trafficking of EGFR is controlled by a novel endosome fusion pathway that is regulated by Rab5 in the absence of PI3K, rather than by the previously defined endosome fusion pathway that is co-regulated by Rab5 and PI3K.     

F-BAR-containing adaptor CIP4 localizes to early endosomes and regulates Epidermal Growth Factor Receptor trafficking and downregulation

Cdc42-Interacting Protein-4 (CIP4) family adaptors have been implicated in promoting Epidermal Growth Factor Receptor (EGFR) internalization, however, their unique or overlapping functions remain unclear. Here, we show that although CIP4 was not required for early events in clathrin-mediated endocytosis of EGFR, CIP4 localizes to vesicles containing EGFR and Rab5. Furthermore, expression of constitutively active Rab5 led to accumulation of CIP4 and the related adaptor Toca-1 in giant endosomes. Using a mutagenesis approach, we show that localization of CIP4 to endosomes is mediated in part via the curved phosphoinositide-binding face of the CIP4 F-BAR domain. Downregulation of CIP4 in A431 epidermoid carcinoma cells by RNA interference led to elevated EGFR levels, compared to control cells. Although surface expression of EGFR was not affected by CIP4 silencing, EGF-induced transit of EGFR from EEA1-positive endosomes to lysosomes was reduced compared to control cells. This correlated with more robust activation of ERK kinase and entry to S phase in CIP4-depleted A431 cells, compared to control cells. The combined silencing of CIP4 and Toca-1 was more effective in driving cells into S phase, suggesting a partial redundancy in their functions. Overall, our results implicate CIP4 and Toca-1 in regulating late events in EGFR trafficking from endosomes that serves to limit sustained ERK activation within the endosomal compartment.     

The PtdIns3P‐Binding Protein Phafin 2 Mediates Epidermal Growth Factor Receptor Degradation by Promoting Endosome Fusion

Phosphatidylinositol 3‐phosphate (PtdIns3P) orchestrates endosomal cargo transport, fusion and motility by recruiting FYVE or PX domain‐containing effector proteins to endosomal membranes. In an attempt to discover novel PtdIns3P effectors involved in the termination of growth factor receptor signalling, we performed an siRNA screen for epidermal growth factor (EGF) degradation, targeting FYVE and PX domain proteins in the human proteome. This screen identified several potential regulators of EGF degradation, including HRS (used as positive control), PX kinase, MTMR4 and Phafin2/PLEKHF2. As Phafin2 has not previously been shown to be required for EGF receptor (EGFR) degradation, we performed further functional studies on this protein. Loss of Phafin2 was found to decrease early endosome size, whereas overexpression of Phafin2 resulted in enlarged endosomes. Moreover, both the EGFR and the fluid‐phase marker dextran were retained in abnormally small endosomes in Phafin2‐depleted cells. In yeast two‐hybrid analysis we identified Phafin2 as a novel interactor of the endosomal‐tethering protein EEA1, and Phafin2 colocalized strongly with EEA1 in microdomains of the endosome membrane. Our results suggest that Phafin2 controls receptor trafficking and fluid‐phase transport through early endosomes by facilitating endosome fusion in concert with EEA1.     

Gαs promotes EEA1 endosome maturation and shuts down proliferative signaling through interaction with GIV (Girdin)

The organization of the endocytic system into biochemically distinct subcompartments allows for spatial and temporal control of the strength and duration of signaling. Recent work has established that Akt cell survival signaling via the epidermal growth factor receptor (EGFR) occurs from APPL early endosomes that mature into early EEA1 endosomes. Less is known about receptor signaling from EEA1 endosomes. We show here that EGF-induced, proliferative signaling occurs from EEA1 endosomes and is regulated by the heterotrimeric G protein Gαs through interaction with the signal transducing protein GIV (also known as Girdin). When Gαs or GIV is depleted, activated EGFR and its adaptors accumulate in EEA1 endosomes, and EGFR signaling is prolonged, EGFR down-regulation is delayed, and cell proliferation is greatly enhanced. Our findings define EEA1 endosomes as major sites for proliferative signaling and establish that Gαs and GIV regulate EEA1 but not APPL endosome maturation and determine the duration and strength of proliferative signaling from this compartment.     

ARAP1 association with CIN85 affects epidermal growth factor receptor endocytic trafficking

Background information. ARAP1 is an Arf (ADP‐ribosylation factor)‐directed GAP (GTPase‐activating protein) that inhibits the trafficking of EGFR (epidermal growth factor receptor) to the early endosome. To further understand the function of ARAP1, we sought to identify proteins that interact with ARAP1. Results. Here we report that ARAP1 associates with the CIN85 (Cbl‐interacting protein of 85 kDa). Arg⁸⁶ and Arg⁹⁰ of ARAP1 and the SH3 (Src homology 3) domains of CIN85 are necessary for the interaction. We found that a mutant of ARAP1 with reduced affinity for CIN85 does not efficiently rescue the effect of reduced ARAP1 expression on EGFR trafficking to the early endosome. Reduced expression of CIN85 has a similar effect as reduced expression of ARAP1 on traffic of the EGFR. Cbl proteins regulate the endocytic trafficking of the EGFR by mediating ubiquitination of the EGFR. Overexpression of ARAP1 reduced ubiquitination of the EGFR by Cbl and slowed Cbl‐dependent degradation of the EGFR. Reduced expression of ARAP1 accelerated degradation of EGFR but did not affect the level of ubiquitination of the receptor that was detected. Conclusion. ARAP1 interaction with CIN85 regulates endocytic trafficking of the EGFR and affects ubiquitination of EGFR. We propose a model in which the ARAP1‐CIN85 complex drives exit of EGF—EGFR–Cbl complex from a pre‐early endosome into a pathway distinct from the early endosome/lysosome pathway.     

APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway.     

Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins

The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural–functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM.