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H Stenmark

Publications and source records attributed to H Stenmark.

At least 19 recordsLinked to original sources
PubMed1999

The endosome fusion regulator early-endosomal autoantigen 1 (EEA1) is a dimer.

EEA1, an early-endosomal protein originally identified as an autoantigen, is essential for endocytic membrane fusion. It interacts with early endosomes via binding to the membrane lipid phosphatidylinositol 3-phosphate (PtdIns3P) and the active form of the small GTPase Rab5. Most of the EEA1 sequence contains heptad repeats characteristic of proteins involved in coiled-coil protein-protein interactions. Here we have investigated the ability of EEA1 to self-interact. Crosslinking of cytosolic and recombinant EEA1 resulted in the disappearance of the 180-kDa monomer in SDS/PAGE and the strong appearance of a approximately 350-kDa crosslinked product. Glycerol gradient centrifugation experiments indicated that native EEA1 had the same hydrodynamic properties as the approximately 350-kDa crosslinked complex. Two-hybrid analysis indicated that N- and C-terminal fragments of EEA1 can interact with themselves, but not with each other, suggesting that EEA1 forms parallel coiled-coil dimers. The ability of the C-terminus of EEA1 to dimerize correlates with its ability to bind to Rab5 and early endosomes, whereas its binding to PtdIns3P is independent of dimerization. These data enable us to propose a model for the quaternary structure of EEA1.

Animals↗
PubMed1998

Cloning of an intracellular protein that binds selectively to mitogenic acidic fibroblast growth factor.

In addition to its extracellular action, there is evidence that acidic fibroblast growth factor (aFGF) acts inside cells. To identify intracellular proteins interacting with aFGF, we screened a HeLa cell library in the yeast two-hybrid system using pLex-aFGF as a bait. A clone binding to aFGF, but not to the non-mitogenic mutant aFGF-K132E, was isolated and characterized. The insert contained an open reading frame corresponding to a novel protein of 42 kDa. The protein, termed aFGF intracellular binding protein (FIBP), is mainly hydrophilic and does not contain an N-terminal signal sequence. In vitro-translated FIBP bound specifically to a fusion protein of maltose-binding protein and aFGF. FIBP became post-translationally associated with microsomes added to the cell-free protein synthesizing system, and the membrane-associated protein bound aFGF with high efficiency. Immunoblots and fluorescence microscopy demonstrated that the protein is present in nuclei and, to a lesser extent, associated with mitochondria and other cytoplasmic membranes. The possibility is discussed that FIBP may be involved in the mitogenic action of aFGF.

Amino Acid Sequence↗
PubMed1998

EEA1 links PI(3)K function to Rab5 regulation of endosome fusion.

GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.

Androstadienes↗
PubMed1998

Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion.

Using the yeast two-hybrid system, we have identified a novel 62 kDa coiled-coil protein that specifically interacts with the GTP-bound form of Rab5, a small GTPase that regulates membrane traffic in the early endocytic pathway. This protein shares 42% sequence identity with Rabaptin-5, a previously identified effector of Rab5, and we therefore named it Rabaptin-5beta. Like Rabaptin-5, Rabaptin-5beta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner. However, Rabaptin-5beta has features that distinguish it from Rabaptin-5. The relative expression levels of the two proteins varies in different cell types. Rabaptin-5beta does not heterodimerize with Rabaptin-5, and forms a distinct complex with Rabex-5, the GDP/GTP exchange factor for Rab5. Immunodepletion of the Rabaptin-5beta complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin-5 complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin-5 complex alone, but maximal fusion efficiency requires the presence of both Rabaptin-5 and Rabaptin-5beta complexes. Our results suggest that Rab5 binds to at least two distinct effectors which cooperate for optimal endocytic membrane docking and fusion.

Amino Acid Sequence↗
PubMed1998

Distinct Rab-binding domains mediate the interaction of Rabaptin-5 with GTP-bound Rab4 and Rab5.

Rabaptin-5 functions as an effector for the small GTPase Rab5, a regulator of endocytosis and early endosome fusion. We have searched for structural determinants that confer functional specificity on Rabaptin-5. Here we report that native cytosolic Rabaptin-5 is present in a homodimeric state and dimerization depends upon the presence of its coiled-coil predicted sequences. A 73 residue C-terminal region of Rabaptin-5 is necessary and sufficient both for the interaction with Rab5 and for Rab5-dependent recruitment of the protein on early endosomes. Surprisingly, we uncovered the presence of an additional Rab-binding domain at the N-terminus of Rabaptin-5. This domain mediates the direct interaction with the GTP-bound form of Rab4, a small GTPase that has been implicated in recycling from early endosomes to the cell surface. Based on these results, we propose that Rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic.

Amino Acid Sequence↗
PubMed1998

Rab17 regulates membrane trafficking through apical recycling endosomes in polarized epithelial cells.

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.

Amino Acid Sequence↗
PubMed1998

Autoantibodies to a novel early endosome antigen 1.

Autoantibodies to EEA1, an antigen on early endosomes, were first reported in the serum of a patient with subacute cutaneous lupus erythematosus (SCLE). Here we have examined 38 sera selected for investigation of autoantibodies to EEA1 on the basis of cytoplasmic vesicle-like reactivity by immunofluorescence. Ten of the sera were reactive to a HeLa cell protein of approximately the same M(r) as human EEA1. Eight of these sera belonged to the IgG1 subclass. Five of the sera reacted with fusion proteins incorporating either the amino (from amino acids 1 to 209) or the carboxyl (incorporating the most C-terminal 300 amino acids) terminus of the human EEA1 protein. Antigens reactive with these 5 sera colocalized with internalized transferrin receptors, indicating their association with early endosomes. The other 5 sera which did not react to both fusion proteins did not colocalize with internalized transferrin receptors. We conclude that 5 of the 38 patients (13%) have autoantibodies to EEA1. None of these patients have SCLE, but have generalized joint pain, polyarthritis, rheumatoid arthritis, or circulating rheumatoid factors.

Aged↗
PubMed1998

Syntaxin-16, a putative Golgi t-SNARE.

Members of the syntaxin family of integral membrane proteins have recently been implicated as vesicle receptors on target membranes, coresponsible for the specificity of intracellular membrane traffic. So far, only a small number of different mammalian syntaxins have been identified. Here we report the cloning of three new human syntaxin cDNAs, presumably originating from alternative splicing of the same transcript. Syntaxin-16A and syntaxin-16B are identical, except that the latter contains an insertion of 21 amino acid residues. Syntaxin-16C is a truncated version of syntaxin-16A, lacking the C-terminal coiled-coil and hydrophobic regions characteristic for syntaxins. Database searches identified putative yeast, plant and nematode homologues of syntaxin-16, indicating that this protein is conserved through evolution, and syntaxin-16 belongs to a new subgroup of syntaxins. Epitope-tagged syntaxin-16A and syntaxin-16B were found to colocalize with the Golgi marker beta-COP, while syntaxin-16C was found in the cytosol. Syntaxin-16A associates posttranslationally with microsomes, and appears to be transported to the Golgi via the endoplasmic reticulum. The three syntaxin-16 forms may have differential roles in intracellular trafficking.

Amino Acid Sequence↗
PubMed1997

A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function.

The small GTPase Rab5 plays an essential role in endocytic traffic. Rab GDP dissociation inhibitor delivers Rab5 to the membrane, where a nucleotide exchange activity allows recruitment of an effector protein, Rabaptin-5. Here we uncovered a novel 60 kDa Rab5-binding protein, Rabex-5. Rabex-5 forms a tight physical complex with Rabaptin-5, and this complex is essential for endocytic membrane fusion. Sequencing of mammalian Rabex-5 by nanoelectrospray mass spectrometry and cloning revealed striking homology to Vps9p, a yeast protein implicated in endocytic traffic. Rabex-5 displays GDP/GTP exchange activity on Rab5 upon delivery of the GTPase to the membrane. This demonstrates that a soluble exchange factor coupled to a Rab effector translocates from cytosol to the membrane, where the complex stabilizes the GTPase in the active state.

Animals↗
PubMed1997

Role of Rab GTPases in membrane traffic.

Small GTPases of the Rab subfamily have been known to be key regulators of intracellular membrane traffic since the late 1980s. Today this protein group amounts to more than 40 members in mammalian cells which localize to distinct membrane compartments and exert functions in different trafficking steps on the biosynthetic and endocytic pathways. Recent studies indicate that cycles of GTP binding and hydrolysis by the Rab proteins are linked to the recruitment of specific effector molecules on cellular membranes, which in turn impact on membrane docking/fusion processes. Different Rabs may, nevertheless, have slightly different principles of action. Studies performed in yeast suggest that connections between the Rabs and the SNARE machinery play a central role in membrane docking/fusion. Further elucidation of this linkage is required in order to fully understand the functional mechanisms of Rab GTPases in membrane traffic.

Animals↗
PubMed1997

Microtubules are involved in bafilomycin A1-induced tubulation and Rab5-dependent vacuolation of early endosomes.

The small GTPase Rab5 is an important regulator of membrane fusion in the early endocytic pathway. Here we have studied at the light microscopy level the morphology of early endosomes in MDCK cells stably expressing a GTPase-deficient Rab5 mutant, Rab5 Q79L, N-terminally tagged with a myc-epitope. These cells contain large vacuoles, readily visible by phase-contrast microscopy. Confocal immunofluorescence microscopy showed the presence of the epitopetagged protein on large perinuclear vacuoles, as well as on smaller peripheral structures. A subset of the perinuclear vacuoles appeared to colocalize with the late endosomal GTPase, Rab7. In addition, a population of very large Rab7-positive, Rab5 Q79L-negative structures were observed, suggesting that an increase in the size of early endosomes may be accompanied by an increased size of later or more mature endocytic structures. Using antibodies against the myc epitope and the early endosomal autoantigen EEA1 as markers, we found that endosomes in wild-type and mutant MDCK cells rapidly tubulate in the presence of bafilomycin A1, an inhibitor of vacuolar H(+)-ATPase. Elongated or tubular endosomes partially colocalized with microtubules and were redistributed upon preincubation with the microtubule depolymerizing agent nocodazole before bafilomycin A1 treatment. Treatment of the Rab5 Q79L expressing cells with nocodazole alone led to a spatial redistribution and a significant decrease in the size of EEA1-positive structures, whereas their number increased. These results implicate microtubules in the bafilomycin A1-induced tubulation of endosomes as well as in the vacuolation of endosomes caused by Rab5 Q79L.

Animals↗
PubMed1996

Endosomal localization of the autoantigen EEA1 is mediated by a zinc-binding FYVE finger.

EEA1, a 162-kDa autoantigen associated with subacute cutaneous systemic lupus erythematosus, is a coiled-coil protein localized to early endosomes and cytosol. At its C terminus, the protein contains a cysteine-rich motif, which is shared with Vps27, Fab1, and Vac1, yeast proteins implicated in membrane traffic (Mu, F. T., Callaghan, J. M., Steele-Mortimer, O., Stenmark, H., Parton, R. G., Campbell, P. L., McCluskey, J., Yeo, J. P., Tock, E. P., and Toh, B. H. (1995) J. Biol. Chem. 270, 13503-13511). Here we show that this motif constitutes a genuine zinc binding domain, which we term the FYVE finger (based on the first letters of four proteins containing this motif). Profile-based data base searches identified the FYVE finger in 11 distinct proteins. The FYVE finger-containing C terminus of EEA1 was found to bind 2 mol equivalents of Zn2+. Mutations of conserved histidine and cysteine residues in the FYVE motif independently reduced zinc binding to 1 mol equivalent. Confocal immunofluorescence microscopy of transfected HEp2 cells revealed that the C-terminal part (residues 1277-1411) of EEA1 colocalizes extensively with a GTPase-deficient mutant of the early endosomal GTPase Rab5, while deletion of the FYVE finger or mutations that interfere with zinc binding cause a cytosolic localization. These results implicate the FYVE finger in the specific localization of EEA1 to endosomes.

Amino Acid Sequence↗
PubMed1995

Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion.

We have identified a novel 100 kDa coiled-coil protein, rabaptin-5, that specifically interacts with the GTP form of the small GTPase Rab5, a potent regulator of endocytic transport. It is mainly cytosolic, but a fraction colocalizes with Rab5 to early endosomes. Expression of a GTPase-deficient Rab5 mutant enhances the binding of rabaptin-5 to enlarged endosomes. Overexpression of rabaptin-5 alone is sufficient to promote expansion of early endosomes. Rab5 recruits rabaptin-5 to purified early endosomes in a GTP-dependent manner, demonstrating functional similarities with other members of the Ras superfamily. Immunodepletion of rabaptin-5 from cytosol strongly inhibits Rab5-dependent early endosome fusion. Rabaptin-5 is thus a Rab effector required for membrane docking and fusion.

Amino Acid Sequence↗
PubMed1995

EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif.

Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly alpha-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine "finger" motifs. The COOH-terminal fingers, Cys-X2-Cys-X12-Cys-X2-Cys and Cys-X2-Cys-X16-Cys-X2-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.

3T3 Cells↗
PubMed1995

Yeast Ypt51p and mammalian Rab5: counterparts with similar function in the early endocytic pathway.

Ypt51p, a small GTPase of Saccharomyces cerevisiae, has been previously identified as a structural homolog of mammalian Rab5. Although disruption analysis revealed that the protein is required for endocytic transport and for vacuolar protein sorting, the precise step controlled by Ypt51p was not determined. In this work we show that by heterologous expression in animal cells Ypt51p was targeted to Rab5-positive early endosomes and stimulated endocytosis. Furthermore, two Ypt51p mutants induced similar morphological alterations as the corresponding Rab5 mutants. Also in yeast cells Ypt51p was found to be required at an early step in endocytic membrane traffic, since alpha-factor accumulated in an early endocytic intermediate in the absence of Ypt51p. Cell fractionation analysis revealed cofractionation of Ypt51p with endocytic intermediates, while no association with the late Golgi compartment could be detected. Indirect immunofluorescence microscopy allowed us to morphologically identify the Ypt51p-containing compartment. Similar to the mammalian system larger Ypt51p-positive structures were revealed upon expression of Ypt51p Q66L. These structures were also positive for alpha-factor receptor and for carboxypeptidase Y, thus providing direct evidence for their endocytic nature and for the convergence of the vacuolar biosynthetic and endocytic pathways.

Animals↗