Jean-Michel Gaullier

The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases. Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known. We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion. Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking. The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1. Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the trans-Golgi network as well. Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate intrans-Golgi network to endosome as well as in endocytic membrane traffic. The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases. Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known. We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion. Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking. The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1. Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the trans-Golgi network as well. Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate intrans-Golgi network to endosome as well as in endocytic membrane traffic. solubleN-ethylmaleimide-sensitive factor attachment protein receptor glutathione S-transferase maltose-binding protein trans-Golgi network phosphate-buffered saline polyacrylamide gel electrophoresis phosphatidylinositol 3-phosphate dithiothreitol guanosine 5′-3-O-(thio)triphosphate The delivery of transport vesicles to their correct destination membrane is ensured by a complex molecular machinery. The SNARE1 and Rab protein families have been assigned a special role in vesicle targeting (1Pfeffer S.R. Nat. Cell Biol. 1999; 1: E17-E22Crossref PubMed Scopus (360) Google Scholar). SNARE proteins on vesicles form tight complexes with their complementary SNARE proteins on target membranes. This docks the vesicle to the target membrane, and membrane fusion ensues (2Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2004) Google Scholar, 3Ungermann C. Sato K. Wickner W. Nature. 1998; 396: 543-548Crossref PubMed Scopus (279) Google Scholar). Although a large number of SNARE molecules have been detected and localized to distinct intracellular membranes, SNARE pairing does not confer sufficient specificity to vesicle delivery because SNAREs interact rather promiscuously with each other (4Yang B. Gonzalez Jr., L. Prekeris R. Steegmaier M. Advani R.J. Scheller R.H. J. Biol. Chem. 1999; 274: 5649-5653Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Small GTPases of the Rab family appear to add an additional layer of specificity. Like SNAREs, distinct Rab GTPases are localized to distinct membranes and control distinct membrane trafficking routes (5Olkkonen V.M. Stenmark H. Int. Rev. Cytol. 1997; 176: 1-85Crossref PubMed Google Scholar,6Novick P. Zerial M. Curr. Opin. Cell Biol. 1997; 9: 496-504Crossref PubMed Scopus (661) Google Scholar). One of their main functions appears to be the recruitment of proteins that function as tethers prior to SNARE complex formation (1Pfeffer S.R. Nat. Cell Biol. 1999; 1: E17-E22Crossref PubMed Scopus (360) Google Scholar). However, the molecular mechanism that couples Rab-mediated tethering to SNARE complex formation is not known. Homotypic fusion between early endosomes can be readily reconstitutedin vitro and provides a convenient system to examine the role of Rab and SNARE proteins (7Mayorga L.S. Diaz R. Stahl P.D. Science. 1989; 244: 1475-1477Crossref PubMed Scopus (88) Google Scholar, 8Gorvel J.P. Chavrier P. Zerial M. Gruenberg J. Cell. 1991; 64: 915-925Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 9Horiuchi H. Lippé R. McBride H.M. Rubino M. Woodman P. Stenmark H. Rybin V. Wilm M. Ashman K. Mann M. Zerial M. Cell. 1997; 90: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). Such fusion requires the presence of Rab5 on both endosome membranes (10Barbieri M.A. Hoffenberg S. Roberts R. Mukhopadhyay A. Pomrehn A. Dickey B.F. Stahl P.D. J. Biol. Chem. 1998; 273: 25850-25855Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), as well as the Rab5 effector, EEA1 (11Simonsen A. Lippé R. Christoforidis S. Gaullier J.-M. Brech A. Callaghan J. Toh B.-H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (909) Google Scholar). The findings that EEA1 contains two spatially separate Rab5 binding sites, forms rod-shaped coiled-coil dimers, and is required prior to endosomal SNARE function have suggested that it may act as a tethering factor (11Simonsen A. Lippé R. Christoforidis S. Gaullier J.-M. Brech A. Callaghan J. Toh B.-H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (909) Google Scholar, 12Callaghan J. Simonsen A. Gaullier J.-M. Toh B.-H. Stenmark H. Biochem. J. 1999; 338: 539-543Crossref PubMed Scopus (97) Google Scholar, 13Christoforidis S. McBride H.M. Burgoyne R.D. Zerial M. Nature. 1999; 397: 621-626Crossref PubMed Scopus (655) Google Scholar). Here we have investigated if EEA1 is able to interact with SNAREs on early endosomes. Syntaxin-7 was PCR-amplified from a Marathon-Ready human brain cDNA (CLONTECH). cDNAs encoding rat syntaxin-3, rat syntaxin-4, and rat syntaxin-5 were provided by Vesa Olkkonen (National Public Health Institute, Helsinki, Finland), syntaxin-11 by Paul Roche (National Institutes of Health, Bethesda, MD), rat syntaxin-6 by Richard Scheller (Stanford University School of Medicine, Stanford, CA), and human syntaxin-13 by Rohan Teasdale (Monash University Medical School, Melbourne, Australia). Syntaxin-16 constructs were based on syntaxin-16A (14Simonsen A. Bremnes B. Rønning E. Aasland R. Stenmark H. Eur. J. Cell Biol. 1998; 75: 223-231Crossref PubMed Scopus (100) Google Scholar). Yeast two-hybrid bait and prey constructs were obtained by cloning the relevant cDNAs into the polylinker sites of pLexA/pBTM116 (15Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1658) Google Scholar) and pGAD GH (CLONTECH), respectively. For expression of glutathione S-transferase (GST) fusion proteins, pGEX syntaxin-7ΔC and pGEX-syntaxin-16ΔC were obtained by subcloning the respective cDNAs into the polylinker sites of pGEX-5X-3. pGEX-3X-syntaxin-6ΔC was provided by Robert C. Piper (University of Iowa, Iowa City, IA). (Syntaxin-6ΔC, syntaxin-7ΔC, and syntaxin-16ΔC encode amino acids 1–234, 1–217, and 1–279 of the respective proteins.) Myc epitope-tagged constructs were obtained by cloning the respective cDNAs behind the myc epitope of pGEM-myc3 or pGEM-myc4 (14Simonsen A. Bremnes B. Rønning E. Aasland R. Stenmark H. Eur. J. Cell Biol. 1998; 75: 223-231Crossref PubMed Scopus (100) Google Scholar). A human autoimmune serum against EEA1 (16Mu F.T. Callaghan J.M. Steele-Mortimer O. Stenmark H. Parton R.G. Campbell P.L. McCluskey J. Yeo J.P. Tock E.P. Toh B.H. J. Biol. Chem. 1995; 270: 13503-13511Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar) and an affinity purified rabbit anti-EEA1 antibody (11Simonsen A. Lippé R. Christoforidis S. Gaullier J.-M. Brech A. Callaghan J. Toh B.-H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (909) Google Scholar) were used. Mouse monoclonal anti-Myc epitope antibody was from the 9E10 hybridoma (17Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2165) Google Scholar). A mouse monoclonal anti-syntaxin-6 antibody was purchased from Transduction Laboratories. Horseradish peroxidase-conjugated goat antibodies against human, mouse, and rabbit IgG, fluorescein isothiocyanate-labeled goat antibodies against human IgG, and lissamine-rhodamine-labeled goat antibodies against mouse IgG were purchased from Jackson Immunoresearch. The yeast reporter strain L40 (15Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1658) Google Scholar) was co-transformed with the indicated pLexA and pGAD plasmids, and β-galactosidase activities of the transformants were determined as described previously (18Stenmark H. Vitale G. Ullrich O. Zerial M. Cell. 1995; 83: 423-432Abstract Full Text PDF PubMed Scopus (399) Google Scholar). For transient overexpression studies, BHK-21 cells were infected for 1 h with T7 RNA polymerase recombinant vaccinia virus (vT7) and then transfected at 37 °C with pGEM-1 plasmids containing the cDNA of interest, using DOTAP (Boehringer, Mannheim), as described previously (19Stenmark H. Bucci C. Zerial M. Methods Enzymol. 1995; 257: 155-164Crossref PubMed Scopus (35) Google Scholar). The cells were analyzed 6 h post-transfection. Cells on coverslips were fixed with 3% paraformaldehyde, permeabilized with 0.05% Saponin (Sigma) and stained with primary antibodies followed by fluorescein isothiocyanate or lissamine-rhodamine-conjugated secondary antibodies, as described (14Simonsen A. Bremnes B. Rønning E. Aasland R. Stenmark H. Eur. J. Cell Biol. 1998; 75: 223-231Crossref PubMed Scopus (100) Google Scholar). The coverslips were examined with a Leica TCS NT confocal microscope equipped with a Kr/Ar laser. GST and MBP fusion proteins were expressed in Escherichia coli BL-21(DE3) cells (12Callaghan J. Simonsen A. Gaullier J.-M. Toh B.-H. Stenmark H. Biochem. J. 1999; 338: 539-543Crossref PubMed Scopus (97) Google Scholar), whereas recombinant, His6-tagged EEA1 was expressed in insect cells (11Simonsen A. Lippé R. Christoforidis S. Gaullier J.-M. Brech A. Callaghan J. Toh B.-H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (909) Google Scholar). HeLa cells grown in 15-cm dishes were washed in ice-cold PBS, scraped, and homogenized in 400 μl of homogenization buffer (HB) (20 mm Hepes, pH 7.2, 100 mm KCl, 2 mm MgCl2), 1 mm DTT by passage through a 22 gauge needle six times. A post-nuclear supernatant was obtained by centrifugation for 5 min at 6000 rpm. The post-nuclear supernatant was centrifuged at 60,000 rpm for 30 min at 4 °C in a Beckman TLA 100.2 rotor to obtain a cytosolic and a membrane fraction. The membrane fraction was solubilized in HB-1 mm DTT, 1% Triton X-100 (TX-100) containing a mixture of protease inhibitors without EDTA (Roche Molecular Biochemicals) for 45 min on ice before centrifugation at 60,000 rpm for 30 min at 4 °C in a Beckman TLA 100.2 rotor. The cytosol and the soluble membrane fraction were then incubated with 5 μg of recombinant GST, GST-syntaxin-6, GST-syntaxin-7, or GST-syntaxin-16 proteins prebound to glutathione-Sepharose (Amersham Pharmacia Biotech) for 2 h at 4 °C. The beads were subsequently washed four times with ice-cold HB, 0.5% TX-100. EEA1 associated with the beads was detected by SDS-polyacrylamide gel electrophoresis (PAGE), followed by immunoblotting, using a rabbit anti-EEA1 serum and the SuperSignal chemiluminescence kit from Pierce. Aliquots (25 μl) of glutathione-Sepharose beads (Amersham Pharmacia Biotech) were washed three times with HB before incubation with 0.1 nmol of GST or GST-syntaxin-6 for 1 h at room temperature. The beads were then washed three times with HB, 0.5% TX-100 before incubation with 1 μg of recombinant EEA1 proteins (His6-EEA1, MBP-EEA11–209, MBP-EEA11257–1411, and MBP-EEA11257–1411C1405S) in HB, 0.5% TX-100 containing 5 mg/ml bovine serum albumin for 1 h at 4 °C. Finally, the beads were washed four times with HB, 0.5% TX-100. Recombinant EEA1 proteins associated with the beads were detected by SDS-PAGE, followed by immunoblotting with a human anti-EEA1 serum and the SuperSignal chemiluminescence kit from Pierce. In some cases, MBP-EEA11257–1411 was incubated with Rab5-GDP or Rab5-GTPγS prior to addition to the beads. His6-Rab5 (2 mm) (20Stenmark H. Parton R.G. Steele-Mortimer O. Lütcke A. Gruenberg J. Zerial M. EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (768) Google Scholar) was preincubated with 10 mm GDP or GTPγS in 20 mm Hepes, pH 7.2, 100 mmK-acetate, 0.5 mm MgCl2, 2 mm EDTA and 1 mm dithiothreitol for 30 min at 25 °C. The MgCl2 was then adjusted to 15 mm, MBP-EEA11257–1411 (50 nm) was added, and the incubation was continued for 30 min at 25 °C. The mixture was then added to glutathione-Sepharose beads containing 0.1 nmol of GST or GST-syntaxin-6 and left at 4 °C for 60 min. Finally, the beads were washed three times with the same buffer containing 15 mmMgCl2 and 0.05% TX-100, and protein associated with the beads was detected by SDS-PAGE followed by immunoblotting with anti-MBP antibodies (11Simonsen A. Lippé R. Christoforidis S. Gaullier J.-M. Brech A. Callaghan J. Toh B.-H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (909) Google Scholar). BHK-21 cells grown in 10-cm dishes were transfected as described above. 6 h post transfection, the cells were washes three time with ice-cold PBS and lysed in HB, 1% TX-100 containing a mixture of protease inhibitors without EDTA (Roche Molecular Biochemicals) for 20 min on ice. The lysate was centrifuged at 10,000 rpm for 10 min, and the supernatant was incubated with a human anti-EEA1 serum, with normal human serum, or with 20 μl of anti-Myc-agarose beads (Santa Cruz Biotechnology) at 4 °C for 15 h. Twenty μl of protein G-agarose beads (Santa Cruz Biotechnology) were added to the lysate in the former cases, and incubation was continued for 1 h at 4 °C. The beads were then washed three times with HB, 1% TX-100 and once with PBS. Precipitated proteins were detected by SDS-PAGE, followed by immunoblotting with anti-EEA1 serum or anti-Myc antibody. Because Rab GTPases appear to act upstream of SNARE proteins in membrane docking/fusion, we investigated the possibility that EEA1 may bind to a SNARE molecule as well as to Rab5-GTP. For this purpose, we cloned EEA1 into a yeast two-hybrid “bait” vector and the cytoplasmic domains of various endosomal/trans-Golgi network (TGN) syntaxins (SNAREs) into a “prey” vector. The bait and prey plasmids were cotransformed into a two-hybrid reporter yeast strain, which was subsequently assayed for activation of the reporter gene, lacZ. As shown in TableI, neither syntaxin-7, which is thought to regulate trafficking between endosomes and lysosomes (21Wang H. Frelin L. Pevsner J. Gene ( Amst. ). 1997; 199: PubMed Scopus Google Scholar, T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), which is thought to regulate trafficking between endosomes and the J.P. Roche J. Cell 1999; Google Scholar), which is found in the (14Simonsen A. Bremnes B. Rønning E. Aasland R. Stenmark H. Eur. J. Cell Biol. 1998; 75: 223-231Crossref PubMed Scopus (100) Google Scholar), were found to interact with EEA1 in the two-hybrid In syntaxin-6, which been implicated in trafficking S. J. Scheller R.H. Mol. Biol. Cell. 1997; PubMed Scopus Google Scholar), was found to interact with as indicated by the β-galactosidase associated with the yeast The between EEA1 and syntaxin-6 be EEA1 was cloned into the prey vector and the syntaxins were cloned into the bait although bait constructs in some reporter activation by not We detected between syntaxin-6 and the Rab5 or with Rab5 effector, (18Stenmark H. Vitale G. Ullrich O. Zerial M. Cell. 1995; 83: 423-432Abstract Full Text PDF PubMed Scopus (399) Google H. Parton R.G. Steele-Mortimer O. Lütcke A. Gruenberg J. Zerial M. EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (768) Google Scholar). results indicate that EEA1 and syntaxin-6 interact specifically with each of syntaxin-6 with EEA1 in the yeast two-hybrid reporter yeast cells were with bait constructs in pLexA and prey constructs in β-galactosidase activities of the transformants are as between in a L40 reporter yeast cells were with bait constructs in pLexA and prey constructs in β-galactosidase activities of the transformants are as between the syntaxin-6 of we of EEA1 against syntaxin-6 in the two-hybrid the terminus of EEA1 the C terminus was found to interact with syntaxin-6 We have previously identified as the endosomal binding of EEA1 H. Aasland R. Toh B.H. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), but this with syntaxin-6, that the syntaxin-6 binding is the endosome binding a that interacts with (11Simonsen A. Lippé R. Christoforidis S. Gaullier J.-M. Brech A. Callaghan J. Toh B.-H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (909) Google Scholar), whereas the C terminus of EEA1 a phosphatidylinositol 3-phosphate binding (11Simonsen A. Lippé R. Christoforidis S. Gaullier J.-M. Brech A. Callaghan J. Toh B.-H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (909) Google J. Simonsen A. Gaullier J.-M. Toh B.-H. Stenmark H. Biochem. J. 1999; 338: 539-543Crossref PubMed Scopus (97) Google Scholar, J.-M. Simonsen A. A. Bremnes B. Aasland R. Stenmark H. Nature. 1998; 394: PubMed Scopus Google Scholar). the Rab5 binding the with syntaxin-6 a that C. Sato K. Wickner W. Nature. 1998; 396: 543-548Crossref PubMed Scopus (279) Google and endosome binding H. Aasland R. Toh B.H. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.-M. Simonsen A. A. Bremnes B. Aasland R. Stenmark H. Nature. 1998; 394: PubMed Scopus Google Scholar) to a of syntaxin-6 binding as well. results suggest that the syntaxin-6 binding of EEA1 both the Rab5 binding and the the between EEA1 and syntaxin-6 can be detected we fusion proteins between GST and the cytoplasmic domains of syntaxin-6, syntaxin-7, and GST or the fusion proteins were on glutathione-Sepharose which were incubated with cytosol and membrane from HeLa the we analyzed the by SDS-PAGE and with anti-EEA1 As shown in a of cytosolic and EEA1 to GST-syntaxin-6, whereas EEA1 not with GST GST-syntaxin-7, and We detected with if the between EEA1 and syntaxin-6 is we a GST using recombinant EEA1 of Like cytosolic the recombinant EEA1 was found to bind specifically to GST-syntaxin-6 1 the recombinant C terminus of EEA1 a fusion with binding was found to interact with syntaxin-6, whereas the and the terminus the from the two-hybrid system and indicate that the C terminus of EEA1 interacts directly and specifically with if EEA1 and syntaxin-6 can interact in we to with anti-EEA1 antibodies and by SDS-PAGE and if syntaxin-6 was As shown in 1 in cells (17Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2165) Google Scholar) syntaxin-6 and was with anti-EEA1 but not with a control serum the was EEA1 was found to with anti-Myc from a lysate from cells but not from cells results indicate that EEA1 interacts with syntaxin-6 in as well as in The of binding sites for both Rab5 and syntaxin-6 to the C terminus of EEA1 to if Rab5 and syntaxin-6 bind to EEA1. For this purpose, we GST-syntaxin-6 on glutathione-Sepharose beads and the binding of MBP-EEA11257–1411 in the presence of Rab5 with GDP or the GTPγS the we detected some binding of the EEA1 to GST but the binding to GST-syntaxin-6 was binding was to in the presence of Rab5-GTPγS but in the presence of Rab5-GDP This that the binding sites for Rab5-GTP and syntaxin-6 at the C terminus of EEA1 if EEA1 and syntaxin-6 colocalize in the we cells with antibodies the As found previously (16Mu F.T. Callaghan J.M. Steele-Mortimer O. Stenmark H. Parton R.G. Campbell P.L. McCluskey J. Yeo J.P. Tock E.P. Toh B.H. J. Biol. Chem. 1995; 270: 13503-13511Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar), EEA1 was detected on early endosomes on the other was in a 2 However, syntaxin-6 was detected on and it extensively with EEA1 on early endosomes This is with a that syntaxin-6 present on vesicles and endosomes as well as in the S. J. Scheller R.H. Mol. Biol. Cell. 1997; PubMed Scopus Google Scholar). the of Rab5 is early endosomes because of fusion (20Stenmark H. Parton R.G. Steele-Mortimer O. Lütcke A. Gruenberg J. Zerial M. EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (768) Google Scholar). if syntaxin-6 is endosomes, we transfected cells with and stained with anti-EEA1 and anti-syntaxin-6 The large endosomes EEA1 2 as well as syntaxin-6 2 and the two proteins on 2 a of epitope-tagged syntaxins were with syntaxin-6, syntaxin-7, and syntaxin-13 were found to be on the early endosomes, whereas syntaxin-4, and were not not results indicate that syntaxin-6 with EEA1 on endosomes, and that syntaxin-6 a of syntaxins that can be found on The between syntaxin-6 and EEA1 is with the that two proteins may interact to regulate trafficking. This provides the for a between a Rab effector and a SNARE a between the Rab and the SNARE been detected in However, this not been to be Science. 1997; PubMed Scopus Google Scholar). yeast recently been shown to interact directly with the Rab5 as well as with a of the family of SNARE Curr. Biol. 1999; 9: Full Text Full Text PDF PubMed Scopus Google Scholar, H. B.F. Mol. Biol. Cell. 1999; PubMed Scopus Google Scholar). and have previously been found in a complex with M. Mol. Biol. Cell. 1997; PubMed Scopus Google Scholar), a yeast of syntaxin-6 Scheller R.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). been implicated in the of vesicles with early endosomes Curr. Biol. 1999; 9: Full Text Full Text PDF PubMed Scopus Google Scholar, M. Mol. Biol. Cell. 1997; PubMed Scopus Google Scholar, L.S. Wickner W. J. Biol. Chem. Full Text PDF PubMed Google A. H. Mol. Biol. Cell. 1997; PubMed Scopus Google Scholar), and it of with a phosphatidylinositol 3-phosphate binding H. Aasland R. Toh B.H. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This the that may an EEA1 in yeast J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) and that and may regulate trafficking The that syntaxin-6 interacts with a of T.H. Piper J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) is with this We that Rab5 and their yeast and may a role in to early endosome trafficking in addition to endocytic membrane fusion. The between syntaxin-6 and EEA1 the tethering of a vesicle to an early Our that Rab5-GTP and syntaxin-6 bind to the C terminus of EEA1 that syntaxin-6 binding may a between EEA1 and We that SNARE present on the endosome may then with syntaxin-6 to form a tight be to if Rab5 and EEA1 may for the of from the to endosomes. it be to if syntaxin-6 may regulate early endosome fusion. The of endosomal SNAREs the possibility that EEA1 may interact with other SNAREs to regulate this We Rønning for and Vesa Robert Paul Richard and Rohan Teasdale for

Human colon adenocarcinoma cells (WiDr) and Chinese hamster lung fibroblasts cells (V79) were incubated with different concentrations of 5-aminolevulinic acid (ALA), and the production of protoporphyrin IX (PpIX) was studied using several techniques. The amount of PpIX produced per cell increased with increasing ALA concentration according to different kinetics for the 2 cell lines. For both cell lines a cell density dependency of the PpIX synthesis was observed. For saturating ALA concentrations, 2-3 times more PpIX was produced per cell at a density of 5 x 10(4) than at a density of 5 x 10(3) cells/cm2. The photosensitivity of cells appeared to increase even more than the PpIX content, indicating a cooperative effect in inactivation. The PpIX production rate increased with cell size and was about 1.9 times higher for cells in the G2 + M phase than for cells in the G1 phase of the cell cycle. Neither cell size nor cell cycle distribution were significantly dependent on cell density.

PURPOSE: Cytarabine (ara-C) has limited activity in solid tumours. CP-4055 (ELACYT) is a novel ara-C-5'-elaidic acid ester that may circumvent this limitation. CP-4055 maximum tolerated dose (MTD), pharmacokinetics and antitumor activity have been investigated in patients with solid tumours. MATERIAL AND METHODS: Thirty-four patients (19 malignant melanoma, 8 ovarian cancers and 7 NSCLC) received CP-4055 as a 30 min, or 2 hr intravenous (IV) infusion daily for 5 consecutive days every 3 or 4 weeks (D1-5 q3w or D1-5 q4w) in a dose escalation designed study with doses ranging from 30 to 240 mg/m(2)/day. RESULTS: The most frequent CTC grade 1-2 adverse events (AEs) were nausea, fatigue, vomiting, anorexia and pyrexia. Most of the grade 3-4 AEs were neutropenia. The MTD was 200 mg/m(2)/day and 240 mg/m(2)/day for D1-5 q3w and D1-5 q4w, respectively. The MTD was independent of infusion time in the 4 week schedule. CP-4055 was maintained in plasma for up to 5-10 hr at dose levels >150 mg/m(2)/day. One objective partial response (PR) with time to progression (TTP) of 22 months was reported in an advanced malignant melanoma patient. CONCLUSION: CP-4055 was well tolerated; the majority of the AEs were of CTC grade 1. The 3 week schedule was not recommended due to neutropenic nadir between days 18-26. The recommended dose was 200 mg/m(2)/day in a D1-5 q4w schedule. Efficacy data suggest that CP-4055 might be active in treatment of solid tumours.