Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane traf®cking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind speci®cally, and with high af®nity, to PI(3)P both in vitro and in vivo. When expressed in ®broblasts, a tagged probe localized to endosomes, as detected by¯uorescence microscopy. Electron microscopy of untransfected ®broblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells de®cient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27D yeast cells, which have impaired endosome to vacuole traf®cking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in de®ning the early endosome and the subsequent steps leading to multivesicular endosome formation.

After endocytosis, some membrane proteins recycle from early endosomes to the plasma membrane whereas others are transported to late endosomes and lysosomes for degradation. Conjugation with the small polypeptide ubiquitin is a signal for lysosomal sorting. Here we show that the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, is involved in the endosomal sorting of ubiquitinated membrane proteins. Hrs contains a clathrin-binding domain, and by electron microscopy we show that Hrs localizes to flat clathrin lattices on early endosomes. We demonstrate that Hrs binds directly to ubiquitin by way of a ubiquitin-interacting motif (UIM), and that ubiquitinated proteins localize specifically to Hrs- and clathrin-containing microdomains. Whereas endocytosed transferrin receptors fail to colocalize with Hrs and rapidly recycle to the cell surface, transferrin receptors that are fused to ubiquitin interact with Hrs, localize to Hrs- and clathrin-containing microdomains and are sorted to the degradative pathway. Overexpression of Hrs strongly and specifically inhibits recycling of ubiquitinated transferrin receptors by a mechanism that requires a functional UIM. We conclude that Hrs sorts ubiquitinated membrane proteins into clathrin-coated microdomains of early endosomes, thereby preventing their recycling to the cell surface.

Phosphatidylinositol-3-phosphate [PtdIns(3)P] regulates endocytic and autophagic membrane traffic. In order to understand the downstream effects of PtdIns(3)P in these processes, it is important to identify PtdIns(3)P-binding proteins, many of which contain FYVE zinc-finger domains. Here, we describe a novel giant FYVE-domaincontaining protein, named autophagy-linked FYVE protein (Alfy). Alfy is ubiquitously expressed, shares sequence similarity with the Chediak-Higashi-syndrome protein and has putative homologues in flies, nematodes and fission yeast. Alfy binds PtdIns(3)P in vitro and partially colocalizes with PtdIns(3)P in vivo. Unlike most other FYVE-domain proteins, Alfy is not found on endosomes but instead localizes mainly to the nuclear envelope. When HeLa cells are starved or treated with a proteasome inhibitor, Alfy relocalizes to characteristic filamentous cytoplasmic structures located close to autophagic membranes and ubiquitin-containing protein aggregates. By electron microscopy, similar structures can be found within autophagosomes. We propose that Alfy might target cytosolic protein aggregates for autophagic degradation.

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. We found a kinesin-3, KIF16B, that transports early endosomes to the plus end of microtubules in a process regulated by the small GTPase Rab5 and its effector, the phosphatidylinositol-3-OH kinase hVPS34. In vivo, KIF16B overexpression relocated early endosomes to the cell periphery and inhibited transport to the degradative pathway. Conversely, expression of dominant-negative mutants or ablation of KIF16B by RNAi caused the clustering of early endosomes to the perinuclear region, delayed receptor recycling to the plasma membrane, and accelerated degradation. These results suggest that KIF16B, by regulating the plus end motility of early endosomes, modulates the intracellular localization of early endosomes and the balance between receptor recycling and degradation. We propose that this mechanism could have important implications for signaling.

Aggregation of receptors specific for the constant region of immunoglobulin G activates a repertoire of monocyte responses that can lead ultimately to targeted cell killing via antibody-directed cellular cytotoxicity. The high affinity receptor, Fc␥RI, contains no recognized signaling motif in its cytoplasmic tail but rather utilizes the ␥-chain of Fc⑀RI as an accessory molecule to recruit tyrosine kinases for signal transduction. We show here that, in a human monocytic cell line primed with interferon-␥, Fc␥RI mobilizes intracellular calcium stores using a novel pathway that involves tyrosine kinase coupling to phospholipase D and resultant downstream activation of sphingosine kinase. Moreover, Fc␥RI is not coupled to phospholipase C; hence, calcium release from intracellular stores occurred in the absence of any measurable rise in inositol triphosphate. Finally, as this novel activation pathway is also shown to be responsible for mediating the vesicular trafficking of internalized immune complexes for degradation, it is likely to play a key role in controlling intracellular events triggered by Fc␥RI.

FYVE zinc finger domains, which are conserved in multiple proteins from yeast to man, interact specifically with the membrane lipid phosphatidylinositol 3-phosphate (PtdIns(3)P). Here we have investigated the structural requirements for the interaction of the FYVE finger of the early endosome antigen EEA1 with PtdIns(3)P and early endosomes. The binding of the FYVE finger to PtdIns(3)P is Zn 2؉ -dependent, and Zn 2؉could not be replaced by any other bivalent cations tested. By surface plasmon resonance, the wild-type FYVE finger was found to bind to PtdIns(3)P with an apparent K D of about 50 nM and a 1:1 stoichiometry. Mutagenesis of cysteines involved in Zn 2؉ coordination, basic residues thought to be directly involved in ligand binding and other conserved residues, resulted in a 6-to >100-fold decreased affinity for PtdIns(3)P. A mutation in the putative PtdIns(3)P-binding pocket, R1375A, may prove particularly informative, because it led to a strongly decreased affinity for PtdIns(3)P without affecting the FYVE three-dimensional structure, as measured by fluorescence spectroscopy. Whereas the C terminus of EEA1 localizes to early endosomes when expressed in mammalian cells, all the FYVE mutants with reduced affinity for PtdIns(3)P were found to be largely cytosolic. Furthermore, whereas expression of the wild-type EEA1 C terminus interferes with early endosome morphology, the point mutants were without detectable effect. These results support recently proposed models for the ligand binding of the FYVE domain and indicate that PtdIns(3)P binding is crucial for the localization and function of EEA1.Phosphatidylinositol 3-kinases are important regulators of vital cellular processes, including endocytic membrane trafficking, signal transduction, apoptosis, and cytoskeletal organization (1-3). The recent discovery of the conserved FYVE zinc finger (for Fab1p, YOTB, Vac1p, EEA1) 1 (4) as a phosphatidylinositol 3-phosphate (PtdIns (3)P) -specific domain has shed light on the function of this phosphatidylinositol 3-kinase product in cellular processes (5-8). FYVE finger proteins include the membrane trafficking regulators EEA1, Hrs, Vac1p, Vps27p, and Fab1p, the signal transducer SARA, the putative cytoskeletal regulator Fgd1, as well as a number of proteins with unknown function (5, 9 -15).The determination of the crystal structure of the ligand-free FYVE domain of the yeast vacuolar sorting protein Vps27p at high resolution has enabled the modeling of the FYVE-PtdIns (3)P interaction (16). According to this model, the basic residues present in the ␤1 strand ( Fig.

Transforming growth factor ␤ (TGF␤) receptors require SARA for phosphorylation of the downstream transducing Smad proteins. SARA, a FYVE finger protein, binds to membrane lipids suggesting that activated receptors may interact with downstream signaling molecules at discrete endocytic locations. In the present study, we reveal a critical role for the early endocytic compartment in regulating Smad-dependent signaling. Not only is SARA localized on early endosomes, but also its minimal FYVE finger sequence is sufficient for early endosomal targeting. Expression of a SARA mutant protein lacking the FYVE finger inhibits downstream activin A signaling in endothelial cells. Moreover, a dominant-negative mutant of Rab5, a crucial protein for early endosome dynamics, causes phosphorylation and nuclear translocation of Smads leading to constitutive (i.e. ligand independent) transcriptional activation of a Smad-dependent promoter in endothelial cells. As inhibition of endocytosis using the K44A negative mutant of dynamin and RN-tre did not lead to activation of Smaddependent transcription, the effects of the dominantnegative Rab5 are likely to be a consequence of altered membrane trafficking of constitutively formed TGF␤/ activin type I/II receptor complexes at the level of early endosomes. The results suggest an important interconnection between early endosomal dynamics and TGF␤/ activin signal transduction pathways.The transforming growth factor ␤ (TGF␤) 1 superfamily is a large group of secreted polypeptide growth factors, which include the TGF␤s, the activins, and the bone morphogenetic proteins. Members of this family play critical roles during embryogenesis and in maintaining tissue homeostasis in adult life. Deregulated TGF␤ family signaling has been implicated in multiple developmental disorders and in various human diseases, including cancer (1). Some of these disorders, such as hereditary hemorrhagic teleangiectasia and primary pulmonary hypertension, involve altered TGF␤ family signaling regulating vasculogenic and angiogenic responses of endothelial cells (2-6). Indeed, TGF␤1 is known to influence both endothelial cell proliferation and critical endothelial cell-pericyte interactions occurring during vessel maturation (7). We have recently shown also that activin A affects endothelial cell function leading to inhibition of angiogenesis and decreased vessel wall integrity (8).The TGF␤/activin family members signal through heteromeric complexes of transmembrane type I and type II serinethreonine kinase receptors. The type II receptor kinase phosphorylates the type I receptor kinase which in turn phosphorylates the downstream transducer proteins, Smad2 and Smad3 (reviewed in Ref. 9). The latter associate with Smad4 and the resulting complex translocates to the nucleus, where they control transcription of target genes. Recent data show that, in the case of the TGF␤ receptor and most likely in the case of activin (10), the Smad-binding protein SARA plays an important role in phosphorylation of Smad2 and Smad3 by TG...

PtdIns3P is a phosphoinositide 3-kinase product that has been strongly implicated in regulating membrane trafficking in both mammalian and yeast cells. PtdIns3P has been shown to be specifically located on membranes associated with the endocytic pathway. Proteins that contain FYVE zinc-finger domains are recruited to PtdIns3P-containing membranes. Structural information is now available concerning the interaction between FYVE domains and PtdIns3P. A number of proteins have been identified which contain a FYVE domain, and in this review we discuss the functions of PtdIns3P and its FYVE-domain-containing effector proteins in membrane trafficking, cytoskeletal regulation and receptor signalling.