Peter van der Sluijs

The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway

rab4 is a ras-like GTP-binding protein that associates with early endosomes in a cell cycle-dependent fashion. To determine its role during endocytosis, we generated stable cell lines that overexpressed mutant or wild-type rab4. By measuring endocytosis, transport to lysosomes, and recycling, we found that overexpression of wild-type rab4 had differential effects on the endocytic pathway. Although initial rates of internalization and degradation were not inhibited, the transfectants exhibited a 3-fold decrease in fluid phase endocytosis as well as an alteration in transferrin receptor (Tfn-R) recycling. Wild-type rab4 caused a redistribution of Tfn-Rs from endosomes to the plasma membrane. It also blocked iron discharge by preventing the delivery of Tfn to acidic early endosomes, instead causing Tfn accumulation in a population of nonacidic vesicles and tubules. rab4 thus appears to control the function or formation of endosomes involved in recycling.

PDGF-regulated rab4-dependent recycling of αvβ3 integrin from early endosomes is necessary for cell adhesion and spreading

Abstract Background: It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. Results: The endo/exocytic cycle of αvβ3 and α5β1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, αvβ3 integrin, but not α5β1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed αvβ3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated αvβ3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for αvβ3), but adhesion to fibronectin (a ligand for α5β1 and αvβ3) was unchanged. Conclusions: We propose that growth factor-regulated, rab4-dependent recycling of αvβ3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7.

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes.

Short-chain ubiquitination mediates the regulated endocytosis of the aquaporin-2 water channel

To regulate mammalian water homeostasis, arginine-vasopressin (AVP) induces phosphorylation and thereby redistribution of renal aquaporin-2 (AQP2) water channels from vesicles to the apical membrane. Vice versa, AVP (or forskolin) removal and hormones activating PKC cause AQP2 internalization, but the mechanism is unknown. Here, we show that a fraction of AQP2 is modified with two to three ubiquitin moieties in vitro and in vivo. Mutagenesis revealed that AQP2 is ubiquitinated with one K63-linked chain at K270 only. In Madin–Darby canine kidney cells, AQP2 ubiquitination occurs preferentially when present in the apical membrane, is transiently increased with forskolin removal or PKC activation, and precedes its internalization. Internalization kinetics assays with wild type (wt) and ubiquitination-deficient (K270R) AQP2 revealed that ubiquitination enhances AQP2 endocytosis. Electron microscopy showed that a translational fusion of AQP2 with ubiquitin (AQP2-Ub) localized particularly to internal vesicles of multivesicular bodies (MVBs), whereas AQP2-K270R largely localized to the apical membrane, early endosomes, and the limiting membrane of MVBs. Consistent with this distribution pattern, lysosomal degradation was extensive for AQP2-Ub, low for AQP2-K270R, and intermediate for wt-AQP2. Our data show that short-chain ubiquitination is involved in the regulated endocytosis, MVB sorting, and degradation of AQP2 and may be the mechanism used by AVP removal and PKC-activating hormones to reduce renal water reabsorption. Moreover, because several other channels are also (short-chain) ubiquitinated, our data suggest that ubiquitination may be a general mediator for the regulated endocytosis and degradation of channels in higher eukaryotes.

Regulation of membrane transport through the endocytic pathway by rabGTPases

Small GTP binding proteins of the rab family are associated with the cytoplasmic surface of compartments of the central vacuolar system. Several of them, including rab5, rab4 and rab11, are localized to early endocytic organelles where they regulate distinct events in the transferrin receptor pathway. Whereas rab5 is controlling transport to early endosomes, rab4 and rab11 are involved in the regulation of recycling back to the plasma membrane. How GTP-hydrolysis of rab bound GTP is related to the role of these proteins in endocytosis is not yet known, but quick progress is being made towards this goal through the identification of proteins regulating the activity of these rab proteins.

Protein kinase B/Akt acts via glycogen synthase kinase 3 to regulate recycling of αvβ3 and α5β1 integrins

ABSTRACT Protein kinase B (PKB)/Akt is known to promote cell migration, and this may contribute to the enhanced invasiveness of malignant cells. To elucidate potential mechanisms by which PKB/Akt promotes the migration phenotype, we have investigated its role in the endosomal transport and recycling of integrins. Whereas the internalization of αvβ3 and α5β1 integrins and their transport to the recycling compartment were independent of PKB/Akt, the return of these integrins (but not internalized transferrin) to the plasma membrane was regulated by phosphatidylinositol 3-kinases and PKB/Akt. The blockade of integrin recycling and cell spreading on integrin ligands effected by inhibition of PKB/Akt was reversed by inhibition of glycogen synthase kinase 3 (GSK-3). Moreover, expression of nonphosphorylatable active GSK-3β mutant GSK-3β-A9 suppressed recycling of α5β1 and αvβ3 and reduced cell spreading on ligands for these integrins, indicating that PKB/Akt promotes integrin recycling by phosphorylating and inactivating GSK-3. We propose that the ability of PKB/Akt to act via GSK-3 to promote the recycling of matrix receptors represents a key mechanism whereby integrin function and cell migration can be regulated by growth factors.

Glycosylation Is Important for Cell Surface Expression of the Water Channel Aquaporin-2 but Is Not Essential for Tetramerization in the Endoplasmic Reticulum

Aquaporin-2 (AQP2) is a pore-forming protein that is required for regulated reabsorption of water from urine. Mutations in AQP2 lead to nephrogenic diabetes insipidus, a disorder in which functional AQP2 is not expressed on the apical cell surface of kidney collecting duct principal cells. The mechanisms and pathways directing AQP2 from the endoplasmic reticulum to the Golgi complex and beyond have not been defined. We found that ∼25% of newly synthesized AQP2 is glycosylated. Nonglycosylated and complex-glycosylated wild-type AQP2 are stable proteins with a half-life of 6-12 h and are both detectable on the cell surface. We show that AQP2 forms tetramers in the endoplasmic reticulum during or very early after synthesis and reaches the Golgi complex in 1-1.5 h. We also report that glycosylation is neither essential for tetramerization nor for transport from the endoplasmic reticulum to the Golgi complex. Instead, the N-linked glycan is important for exit from the Golgi complex and sorting of AQP2 to the plasma membrane. These results are important for understanding the molecular mechanisms responsible for the intracellular retention of AQP2 in nephrogenic diabetes insipidus.

Regulation of Membrane Transport by rab GTPases

Membrane flow through the cell is a highly dynamic process in which intracellular compartments communicate via tubulo-vesicular structures shuttling cargo molecules to their destinations. Transport carriers are formed at a donor compartment and navigate through the cytoplasm to the target organelle, on which they subsequently dock and fuse. Many of these events are regulated by the cooperative action of monomeric rab GTPases and their effector proteins. Research in recent years resulted in the identification of many rab effectors, providing first glimpses how the GTPase switch of individual rab proteins is utilized in discrete transport steps.

Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex

A;lthough glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.

Rab6, Rab8, and MICAL3 cooperate in controlling docking and fusion of exocytotic carriers.

Rab6 is a conserved small GTPase that localizes to the Golgi apparatus and cytoplasmic vesicles and controls transport and fusion of secretory carriers [1]. Another Rab implicated in trafficking from the trans-Golgi to the plasma membrane is Rab8 [2-5]. Here we show that Rab8A stably associates with exocytotic vesicles in a Rab6-dependent manner. Rab8A function is not needed for budding or motility of exocytotic carriers but is required for their docking and fusion. These processes also depend on the Rab6-interacting cortical factor ELKS [1], suggesting that Rab8A and ELKS act in the same pathway. We show that Rab8A and ELKS can be linked by MICAL3, a member of the MICAL family of flavoprotein monooxygenases [6]. Expression of a MICAL3 mutant with an inactive monooxygenase domain resulted in a strong accumulation of secretory vesicles that were docked at the cell cortex but failed to fuse with the plasma membrane, an effect that correlated with the strongly reduced mobility of MICAL3. We propose that the monooxygenase activity of MICAL3 is required to regulate its own turnover and the concomitant remodeling of vesicle-docking protein complexes in which it is engaged. Taken together, the results of our study illustrate cooperation of two Rab proteins in constitutive exocytosis and implicates a redox enzyme in this process.