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The Pleckstrin Homology Domains of Dynamin Isoforms Require Oligomerization for High Affinity Phosphoinositide Binding

1998; Elsevier BV; Volume: 273; Issue: 42 Linguagem: Inglês

10.1074/jbc.273.42.27725

ISSN

1083-351X

Autores

Daryl E. Klein, Anthony Lee, David W. Frank, Michael S. Marks, Mark A. Lemmon,

Tópico(s)

Lipid Membrane Structure and Behavior

Resumo

The dynamins are 100-kDa GTPases involved in the scission event required for formation of endocytotic vesicles. The two main described mammalian dynamins (dynamin−1 and dynamin−2) both contain a pleckstrin homology (PH) domain, which has been implicated in dynamin binding to (and activation by) acidic phospholipids, most notably phosphoinositides. We demonstrate that the PH domains of both dynamin isoforms require oligomerization for high affinity phosphoinositide binding. Strong phosphoinositide binding was detected only when the PH domains were dimerized by fusion to glutathioneS-transferase, or via a single engineered intermolecular disulfide bond. Phosphoinositide binding specificities agreed reasonably with reported effects of different phospholipids on dynamin GTPase activity. Although they differ in their ability to inhibit rapid endocytosis in adrenal chromaffin cells, the dynamin−1 and dynamin−2 PH domains showed identical phosphoinositide binding specificities. Since oligomerization is required for binding of the dynamin PH domain to phosphoinositides, it follows that PH domain-mediated phosphoinositide binding will favor oligomerization of intact dynamin (which has an inherent tendency to self-associate). We propose that the dynamin PH domain thus mediates the observed cooperative binding of dynamin to membranes containing acidic phospholipids and promotes the self-assembly that is critical for both stimulation of its GTPase activity and its ability to achieve membrane scission. The dynamins are 100-kDa GTPases involved in the scission event required for formation of endocytotic vesicles. The two main described mammalian dynamins (dynamin−1 and dynamin−2) both contain a pleckstrin homology (PH) domain, which has been implicated in dynamin binding to (and activation by) acidic phospholipids, most notably phosphoinositides. We demonstrate that the PH domains of both dynamin isoforms require oligomerization for high affinity phosphoinositide binding. Strong phosphoinositide binding was detected only when the PH domains were dimerized by fusion to glutathioneS-transferase, or via a single engineered intermolecular disulfide bond. Phosphoinositide binding specificities agreed reasonably with reported effects of different phospholipids on dynamin GTPase activity. Although they differ in their ability to inhibit rapid endocytosis in adrenal chromaffin cells, the dynamin−1 and dynamin−2 PH domains showed identical phosphoinositide binding specificities. Since oligomerization is required for binding of the dynamin PH domain to phosphoinositides, it follows that PH domain-mediated phosphoinositide binding will favor oligomerization of intact dynamin (which has an inherent tendency to self-associate). We propose that the dynamin PH domain thus mediates the observed cooperative binding of dynamin to membranes containing acidic phospholipids and promotes the self-assembly that is critical for both stimulation of its GTPase activity and its ability to achieve membrane scission. guanosine 5′-O-(thiotriphosphate) pleckstrin homology rapid endocytosis 5)P2, phosphatidylinositol-4,5-bisphosphate 4,5)P3, inositol-1,4,5-trisphosphate dynamin−1 dynamin−2 dynamin−1 PH domain dynamin−2 PH domain phospholipase-C, PtdCho, phosphatidylcholine phosphatidylserine glutathione S-transferase dithiothreitol tris(2-carboxyethyl phosphine) hydrochloride small unilamellar vesicle proline/arginine-rich domain. The dynamins are GTPases of 100 kDa that play a key role in the scission event leading to endocytotic vesicle formation (1Warnock D.E. Schmid S.L. BioEssays. 1996; 18: 885-893Crossref PubMed Scopus (137) Google Scholar, 2Damke H FEBS Lett. 1996; 389: 48-51Crossref PubMed Scopus (63) Google Scholar, 3Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (256) Google Scholar). In cells expressing mutated dynamin−1 defective in GTP binding and hydrolysis (4Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (398) Google Scholar, 5van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.M. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (591) Google Scholar, 6Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1046) Google Scholar), receptor-mediated endocytosis is inhibited, and invaginated coated pits accumulate, on which dynamin appears uniformly distributed (6Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1046) Google Scholar). If GTP hydrolysis (but not binding) by dynamin is inhibited in synaptosomes with GTPγS,1 constricted coated pits accumulate, in which dynamin forms a "collar" around the constriction (7Takei K. McPherson P.S. Schmid S.L. De Camilli P. Nature. 1995; 374: 186-190Crossref PubMed Scopus (657) Google Scholar). A similar collar occurs in shibire Drosophila, which have a mutated dynamin homolog (8Kosaka T. Ikeda K. J. Neurobiol. 1983; 14: 207-225Crossref PubMed Scopus (254) Google Scholar). A model has thus emerged for the role of dynamin in receptor-mediated endocytosis (1Warnock D.E. Schmid S.L. BioEssays. 1996; 18: 885-893Crossref PubMed Scopus (137) Google Scholar, 2Damke H FEBS Lett. 1996; 389: 48-51Crossref PubMed Scopus (63) Google Scholar) in which it is first targeted to clathrin-coated pits in a GDP-bound (or nucleotide-free) form. Upon GTP binding, dynamin is proposed to self-assemble at the necks of invaginated coated pits to form collars, and GTP hydrolysis by dynamin in the collars is finally thought to pinch off the endocytotic vesicle. Three mammalian dynamin isoforms are known (1Warnock D.E. Schmid S.L. BioEssays. 1996; 18: 885-893Crossref PubMed Scopus (137) Google Scholar, 3Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (256) Google Scholar, 9Vallee R.B. Okamato P.M. Trends Cell Biol. 1995; 5: 43-47Abstract Full Text PDF PubMed Scopus (50) Google Scholar). Dynamin−1 is expressed only in neurons (10Scaife R. Margolis R.L. J. Cell Biol. 1990; 111: 3023-3033Crossref PubMed Scopus (82) Google Scholar, 11Sontag J.M. Fykse E.M. Ushkaryov Y. Liu J.P. Robinson P.J. Südhof T.C. J. Biol. Chem. 1994; 269: 4547-4554Abstract Full Text PDF PubMed Google Scholar), dynamin−2 is ubiquitously expressed (11Sontag J.M. Fykse E.M. Ushkaryov Y. Liu J.P. Robinson P.J. Südhof T.C. J. Biol. Chem. 1994; 269: 4547-4554Abstract Full Text PDF PubMed Google Scholar, 12Cook T.A. Urrutia R. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 644-648Crossref PubMed Scopus (163) Google Scholar), while dynamin−3 is restricted primarily to the testes (13Nakata T. Takemura R. Hirokawa N. J. Cell Sci. 1993; 105: 1-5Crossref PubMed Google Scholar). Each isoform has multiple domains. The N-terminal ≈300 amino acids comprise the GTPase domain, which is followed by two ≈100-amino acid regions of unknown function. A pleckstrin homology (PH) domain extends from residues 521 to 623 (in human dynamin−1), followed by a 130-amino acid domain that interacts with the GTPase domain and acts as a GTPase effector (14Muhlberg A.B. Warnock D.E. Schmid S.L. EMBO J. 1997; 16: 6676-6683Crossref PubMed Scopus (199) Google Scholar). Finally, the C-terminal 100 amino acids form a proline/arginine-rich domain (PRD), which binds in vitro to several SH3 domains (15Gout I. Dhand R. Hiles I.D. Fry M.J. Panayotou G. Das P. Truong O. Totty N.F. Hsuan J. Booker G.W. Cambell I.D. Waterfield M.D. Cell. 1993; 75: 25-36Abstract Full Text PDF PubMed Scopus (485) Google Scholar), and is important in targeting dynamin to coated pits (16Shpetner H.S. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1996; 271: 13-16Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Recent studies have demonstrated a specific role for PRD binding to the amphiphysin SH3 domain in recruiting dynamin to coated pits (17Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (404) Google Scholar, 18Wigge P. Vallis Y. McMahon H.T. Curr. Biol. 1997; 7: 554-560Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). In the absence of membranes, purified dynamin forms a tetramer (14Muhlberg A.B. Warnock D.E. Schmid S.L. EMBO J. 1997; 16: 6676-6683Crossref PubMed Scopus (199) Google Scholar), which further self-assembles into rings or spirals when subjected to low ionic strength conditions (19Hinshaw J. Schmid S.L. Nature. 1995; 374: 190-192Crossref PubMed Scopus (665) Google Scholar) or (at physiological ionic strength) to GDP plus metallofluorides (20Carr J.F. Hinshaw J.E. J. Biol. Chem. 1997; 272: 28030-28035Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The assemblies are morphologically similar to the collars seen in constricted coated vesicles in vivo, and their formation requires the PRD, but not the PH domain (14Muhlberg A.B. Warnock D.E. Schmid S.L. EMBO J. 1997; 16: 6676-6683Crossref PubMed Scopus (199) Google Scholar, 19Hinshaw J. Schmid S.L. Nature. 1995; 374: 190-192Crossref PubMed Scopus (665) Google Scholar). The function of dynamin in endocytosis requires both its self-assembly and GTPase activity. Self-assembly enhances the GTPase activity of dynamin; an effect that can be mimicked in vitroby several multivalent dynamin-binding molecules including microtubules (21Shpetner H.S. Vallee R.B. Nature. 1992; 355: 733-735Crossref PubMed Scopus (171) Google Scholar), glutathione S-transferase (GST)/SH3 domain fusion proteins (e.g. from Grb2) (15Gout I. Dhand R. Hiles I.D. Fry M.J. Panayotou G. Das P. Truong O. Totty N.F. Hsuan J. Booker G.W. Cambell I.D. Waterfield M.D. Cell. 1993; 75: 25-36Abstract Full Text PDF PubMed Scopus (485) Google Scholar), and bivalent antibodies (22Warnock D.E. Terlecky L.J. Schmid S.L. EMBO J. 1995; 14: 1322-1328Crossref PubMed Scopus (76) Google Scholar). The GTPase activity of purified dynamin also shows a cooperative dependence on its concentration (23Tuma P.L. Collins C.A. J. Biol. Chem. 1994; 269: 30842-30847Abstract Full Text PDF PubMed Google Scholar), which correlates with self-assembly (23Tuma P.L. Collins C.A. J. Biol. Chem. 1994; 269: 30842-30847Abstract Full Text PDF PubMed Google Scholar, 24Warnock D.E. Hinshaw J.E. Schmid S.L. J. Biol. Chem. 1996; 271: 22310-22314Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Many activators of dynamin in vitrothus appear to exert their effect simply by enhancing dynamin self-assembly. This is also likely to occur in vivo, although the mechanism of dynamin self-association on coated vesicles is not clearly understood. One class of molecules that enhance both the GTPase activity of dynamin and its self-assembly in vitro is acidic phospholipids, including the phosphoinositides (25Tuma P.L. Stachniak M.C. Collins C.A. J. Biol. Chem. 1993; 268: 17240-17246Abstract Full Text PDF PubMed Google Scholar, 26Tuma P.L. Collins C.A. J. Biol. Chem. 1995; 270: 26707-26714Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) appears to be the most potent (27Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayoyou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (495) Google Scholar), although PtdIns(3,4,5)P3 also has a strong stimulatory effect (28Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), and significant effects are seen with other acidic phospholipids (25Tuma P.L. Stachniak M.C. Collins C.A. J. Biol. Chem. 1993; 268: 17240-17246Abstract Full Text PDF PubMed Google Scholar, 26Tuma P.L. Collins C.A. J. Biol. Chem. 1995; 270: 26707-26714Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 27Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayoyou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (495) Google Scholar, 28Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 29Lin H.C. Barylko B. Achiriloaie M. Albanesi J.P. J. Biol. Chem. 1997; 272: 25999-26004Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Deletion of the PH domain from dynamin-1 abolishes the ability of PtdIns(4,5)P2 to stimulate GTPase activity (27Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayoyou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (495) Google Scholar), while deletion of the PRD has no effect (29Lin H.C. Barylko B. Achiriloaie M. Albanesi J.P. J. Biol. Chem. 1997; 272: 25999-26004Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Thus, as for other PH domains (30Lemmon M.A. Falasca M. Ferguson K.M. Schlessinger J. Trends Cell Biol. 1997; 7: 237-242Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 31Shaw G. BioEssays. 1996; 18: 35-46Crossref PubMed Scopus (254) Google Scholar), there is evidence that phosphoinositide binding to the dynamin PH domain (DynPH) plays a role in its activation. Nonetheless, we have not been able to detect binding of isolated DynPH to any phosphoinositide or inositol phosphate in a variety of assays (32Lemmon M.A. Ferguson K.M. O'Brien R. Sigler P.B. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10472-10476Crossref PubMed Scopus (481) Google Scholar, 33Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (243) Google Scholar), although others have (27Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayoyou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (495) Google Scholar, 34Zheng J. Cahill S.M. Lemmon M.A. Fushman D. Schlessinger J. Cowburn D. J. Mol. Biol. 1996; 255: 14-21Crossref PubMed Scopus (219) Google Scholar) (see below). Furthermore, while the isolated dynamin−1 PH domain (Dyn1PH) inhibits rapid endocytosis (RE) following stimulated catecholamine secretion from adrenal chromaffin cells (35Artalejo C.J. Lemmon M.A. Schlessinger J. Palfrey H.C. EMBO J. 1997; 15: 1565-1574Crossref Scopus (70) Google Scholar), other PH domains that bind strongly to PtdIns(4,5)P2 have no effect. The PH domain from dynamin−2, which shares 81% identity with Dyn1PH, does not affect RE (35Artalejo C.J. Lemmon M.A. Schlessinger J. Palfrey H.C. EMBO J. 1997; 15: 1565-1574Crossref Scopus (70) Google Scholar). Motivated by these observations, we reinvestigated phosphoinositide binding by dynamin PH domains in an effort to understand discrepancies in the literature and to determine whether functional differences (in RE) between Dyn1PH and Dyn2PH can be explained by their phosphoinositide binding specificities or affinities. Dyn1PH has been shown to bind weakly to the PtdIns(4,5)P2head group, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3), in NMR studies, with reported K D values of 4.3 mm (34Zheng J. Cahill S.M. Lemmon M.A. Fushman D. Schlessinger J. Cowburn D. J. Mol. Biol. 1996; 255: 14-21Crossref PubMed Scopus (219) Google Scholar) and 1.2 mm (27Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayoyou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (495) Google Scholar). Zheng et al. (34Zheng J. Cahill S.M. Lemmon M.A. Fushman D. Schlessinger J. Cowburn D. J. Mol. Biol. 1996; 255: 14-21Crossref PubMed Scopus (219) Google Scholar) also found that PtdIns(4,5)P2 and PtdIns-4-P can bind Dyn1PH, but only when detergent-solubilized phospholipids were used, and the final detergent concentration was below critical micelle concentration. In contrast, Salim et al. (27Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayoyou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (495) Google Scholar) found that Dyn1PH binds to PtdIns(4,5)P2-containing vesicles, using a GST fusion protein of Dyn1PH immobilized on a biosensor chip. In our own studies with isolated Dyn1PH, we have been unable to detect significant binding to any phosphoinositide (32Lemmon M.A. Ferguson K.M. O'Brien R. Sigler P.B. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10472-10476Crossref PubMed Scopus (481) Google Scholar, 33Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (243) Google Scholar). In this report, we demonstrate that the PH domains from dynamin−1 and dynamin−2 bind with much higher affinity to phosphoinositides when they are oligomeric; PH domain dimerization was required for detection of significant phosphoinositide binding. Since intact dynamin forms tetramers and higher order assemblies, and this behavior is critical for its physiological function, we suggest that PH domain-mediated binding of dynamin to the membrane surface in vivo requires oligomerization. Furthermore, since Salim et al. (27Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayoyou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (495) Google Scholar) used a (dimeric) GST fusion protein and were able to detect Dyn1PH binding to PtdIns(4,5)P2, our findings provide an explanation for the disagreement between previous studies. Phosphoinositide binding by dimeric dynamin PH domains shows specificity similar to that seen for stimulation of dynamin GTPase activity in vitro (28Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Dyn1PH and Dyn2PH gave identical results, despite their different abilities to inhibit rapid endocytosis in adrenal chromaffin cells (35Artalejo C.J. Lemmon M.A. Schlessinger J. Palfrey H.C. EMBO J. 1997; 15: 1565-1574Crossref Scopus (70) Google Scholar). We suggest that PH domain-mediated binding of dynamin to phosphoinositide-containing membranes can occur only coincident with, or following, its self-assembly. This is likely to explain the observed cooperativity in dynamin binding to membranes, the stabilization of dynamin oligomers by vesicles containing acidic phospholipids (26Tuma P.L. Collins C.A. J. Biol. Chem. 1995; 270: 26707-26714Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), the influence of phosphoinositides on dynamin GTPase activity, and it is likely to have important functional implications. PtdIns-4-P, PtdSer, PtdIns(4,5)P2, and Ins(1,4,5)P3 were from Sigma. Dipalmitoyl PtdIns-3-P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3 were from Matreya (Pleasant Gap, PA). PtdCho and di(dibromostearoyl) PtdCho were from Avanti (Birmingham, AL). Monomeric Dyn1PH and Dyn2PH were produced exactly as described previously (33Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (243) Google Scholar, 35Artalejo C.J. Lemmon M.A. Schlessinger J. Palfrey H.C. EMBO J. 1997; 15: 1565-1574Crossref Scopus (70) Google Scholar). For GST fusion proteins, fragments with the same domain boundaries were subcloned into pGEX-2T and pGEX-2TK (Amersham Pharmacia Biotech), for centrifugation experiments and dot-blot experiments, respectively. For GST fusion protein purification, cells were lysed by sonication in 50 mm Tris, pH 8.0, 150 mm NaCl, containing 1 mm phenylmethylsulfonyl fluoride, 1 mm EDTA, and 1 mm DTT. Protein was bound to glutathione-agarose (Sigma) for 15 min at 4 °C. Beads were washed four times in lysis buffer and once in lysis buffer containing 1 m NaCl, and protein was eluted with 15 mm reduced glutathione. Glutathione was removed by size-exclusion chromatography. Purified GST fusion proteins (pGEX-2TK) were labeled with 32P as described elsewhere (36Margolis B. Young R.A. Glover D.M. Hames B.D. DNA Cloning 2. IRL Press, Oxford, UK1995: 1-14Google Scholar, 37Ron D. Dressler H. BioTechniques. 1992; 13: 866-869PubMed Google Scholar), while bound to glutathione-agarose. Approximately 10 μg of purified protein were incubated (in 75 μl) with 0.75 mCi of [γ-32P]ATP plus 10–20 units of protein kinase A (Sigma) for 30 min at room temperature in 50 mm potassium phosphate, pH 7.15, 10 mm MgCl2, 5 mm NaF, 4.5 mm DTT. After washing extensively with phosphate-buffered saline, containing 1 mm DTT and 1 mm phenylmethylsulfonyl fluoride, 32P-labeled protein was eluted from glutathione-agarose using 15 mmreduced glutathione in phosphate-buffered saline and filtered (0.2 μm) prior to use for dot-blots. Phospholipids at 2 mg/ml in 1:1 chloroform:methanol solution (containing 0.1% HCl) were spotted (2 μl) onto nitrocellulose sheets in the pattern shown in Fig. 1. After drying, nitrocellulose was blocked overnight at 4 °C in Tris-buffered saline plus 3% bovine serum albumin (without detergent). 32P-Labeled GST-PH fusion protein at 0.5 μg/ml in Tris-buffered saline, 3% bovine serum albumin was then used to probe the phosphoinositide-containing nitrocellulose for 30 min at room temperature. Filters were washed five times with Tris-buffered saline (without detergent) and dried, and bound radioactivity was visualized using a PhosphorImager (Molecular Dynamics). The single cysteine in Dyn1PH (Cys607 of human dynamin−1) was mutated to serine using polymerase chain reaction mutagenesis as described previously (35Artalejo C.J. Lemmon M.A. Schlessinger J. Palfrey H.C. EMBO J. 1997; 15: 1565-1574Crossref Scopus (70) Google Scholar). A unique N-terminal cysteine was then introduced by polymerase chain reaction, and the mutated product (CysDyn1PH) was expressed from pET11a in Escherichia coli BL-21 as described previously (33Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (243) Google Scholar). The N terminus of CysDyn1PH has the sequence MCKTSG. DTT was maintained at 5 mm during initial purification steps. Following ion-exchange and ammonium sulfate precipitation, protein was dialyzed overnight into 50 mm sodium phosphate, pH 7.4, containing no DTT. Cu(II) 1,10-phenanthroline was then used to catalyze oxidation for disulfide-mediated dimerization as described elsewhere (38Lee G.F. Lebert M.R. Lilly A.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3391-3395Crossref PubMed Scopus (71) Google Scholar). A mixture containing 60 mmCuSO4 and 200 mm 1,10-phenanthroline (Sigma) in 50 mm sodium phosphate, pH 7.4, was diluted 200-fold into CysDyn1PH at 0.4 mm. Oxidation was allowed to proceed for 1 h at 37 °C and was terminated by addition of EDTA to 10 mm. A comparison of reducing and nonreducing SDS-polyacrylamide gel electrophoresis was used to assess the extent of disulfide-mediated dimerization. In gel filtration (Superose 12), the oxidized protein gave two incompletely resolved peaks corresponding to the dimer and monomer, respectively. Fractions were taken from the beginning of the dimer peak and determined by nonreducing SDS-polyacrylamide gel electrophoresis, analytical ultracentrifugation, and light-scattering measurements to contain >90% PH domain dimer. A similar procedure was followed for Dyn2PH. Sedimentation equilibrium experiments employed the XL-A analytical ultracentrifuge (Beckman). Samples were loaded into six-channel epon charcoal-filled centerpieces, using quartz windows. Experiments were performed at 25 °C using two different speeds (10,000 and 15,000 rpm), detecting at 280 nm, with identical results. Solvent density was taken as 1.003 g/ml, and the partial specific volume of Dyn1PH was estimated from its amino acid composition as 0.734 ml g−1. Experiments were performed with 6 and 15 μm protein, using oxidized CysDyn1PH from the dimer peak obtained in gel filtration chromatography. In each case, experiments were performed both with (1 mm) and without the reducing agent tris(2-carboxyethylphosphine) hydrochloride (TCEP). Data were fit using the Optima XL-A data analysis software (Beckman/MicroCal). Randomly distributed residuals were obtained using fits to a single ideal species, examples of which are shown in Fig. 2. Small unilamellar vesicles (SUVs) were generated by co-dissolving di(dibromostearoyl) phosphatidylcholine (PtdCho) with a single phosphoinositide or phosphatidylserine (PtdSer) at 3% (molar) in 1:1 chloroform:methanol containing 0.1% HCl. Brominated PtdCho was employed to allow efficient pelleting of SUVs by ultracentrifugation (39Tortorella D. London E. Anal. Biochem. 1994; 217: 176-180Crossref PubMed Scopus (15) Google Scholar). Lipid mixtures were dried under nitrogen, followed by high vacuum, and then rehydrated in assay buffer (25 mm HEPES, pH 7.2, 100 mm NaCl) with bath sonication to a total lipid concentration of 25 mm. The pH was adjusted to 7.2, and vesicles were subjected to at least 10 cycles of freeze (liquid N2)-thaw (bath sonication at 45 °C), until optically clear. Centrifugation assay samples (100 μl) contained the PH domain at 10 μm and lipid at total concentrations from 0 to 4 mm, corresponding to 0–120 μmphosphoinositide, or 0–60 μm available phosphoinositide (assuming that 50% is accessible on the SUV outer leaflet). Vesicle/protein mixtures were centrifuged for 1 h at 25 °C at 85,000 rpm in a Beckman Optima TLX ultracentrifuge, using a TLA-120.1 rotor. 75 μl of supernatant were assayed for protein content. After discarding the remaining supernatant, the vesicle pellet was resuspended in 100 μl of buffer by bath sonication, and 75 μl were taken for protein assay. Protein assays employed the Pierce BCA assay, as directed by the manufacturer. For assaying resuspended vesicles, SDS (1%) was added after incubation to remove scattering artifacts. A standard curve for each protein was generated in tandem order to determine the percentage of total protein pelleted. Molar partition coefficients (K), as defined (40Peitsch R.M. McLaughlin S. Biochemistry. 1993; 32: 10436-10443Crossref PubMed Scopus (462) Google Scholar), were estimated by fitting the data (in ORIGIN) to Equation 1.%protein bound=100×K[lipid](1+K[lipid])(Eq. 1) where [lipid] is the concentration of total available lipid (≫[protein]bound), approximated by one-half the total lipid concentration (assuming 50% is available on the SUV outer leaflet), and K is a partition coefficient corresponding to the proportionality constant between the concentration of protein bound to the outer SUV leaflet and its concentration in bulk solution. Determination of K makes no assumptions of stoichiometry, although K D for phosphoinositide binding can be estimated as (mole ratio)/K if 1:1 binding of phosphoinositides is assumed. To analyze phosphoinositide binding by Dyn1PH and Dyn2PH, we first used a simple, qualitative, dot-blot assay. In this assay,32P-labeled GST-PH domain fusions are used to probe nitrocellulose filters spotted with defined quantities of pure phosphoinositides (see "Experimental Procedures"). Dyn1PH and Dyn2PH gave identical results in this screen (Fig. 1), showing significant binding to PtdIns(3,4,5)P3, PtdIns(3,4)P2, and PtdIns-3-P. Having observed binding of both Dyn1PH and Dyn2PH to phosphoinositides in the dot-blot assay, our next aim was to compare their affinities. Our primary motivation was the observation that Dyn1PH, but not Dyn2PH, inhibits RE in adrenal chromaffin cells when introduced as the monomeric protein (35Artalejo C.J. Lemmon M.A. Schlessinger J. Palfrey H.C. EMBO J. 1997; 15: 1565-1574Crossref Scopus (70) Google Scholar). To test whether this functional distinction could be explained by differences in phosphoinositide-binding affinities, we measured the ability of unlabeled Dyn1PH and Dyn2PH (expressed from a pET vector) to compete with 32P-labeled GST-Dyn1PH for binding to PtdIns(3,4)P2 immobilized on small nitrocellulose discs. Surprisingly, no competition by the unlabeled PH domains was seen until they were added at concentrations >100 μm (not shown). Since GST-Dyn1PH is present in these assays at 0.5 μg/ml (≈0.01 μm), this result argues that fusion of Dyn1PH to GST enhances its affinity for the immobilized phosphoinositide by more than 104-fold. Using a gel filtration assay (30Lemmon M.A. Falasca M. Ferguson K.M. Schlessinger J. Trends Cell Biol. 1997; 7: 237-242Abstract Full Text PDF PubMed Scopus (148) Google Scholar) and the PEG-precipitation assay of Fukuda et al. (41Fukuda M. Mikoshiba K. J. Biol. Chem. 1996; 271: 18838-18842Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) (not shown), we found that fusion to GST does not significantly increase binding affinity of the dynamin PH domains for soluble inositol phosphate head groups. K D's for binding of dynamin PH domains to [3H]Ins(1,4,5)P3 or [3H]Ins(1,3,4)P3 were estimated to be in the several hundred micromolar to millimolar range, as reported previously (27Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayoyou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (495) Google Scholar, 34Zheng J. Cahill S.M. Lemmon M.A. Fushman D. Schlessinger J. Cowburn D. J. Mol. Biol. 1996; 255: 14-21Crossref PubMed Scopus (219) Google Scholar), regardless of fusion to GST (not shown). The difference between GST fusion proteins and proteins expressed as free PH domains is therefore not likely to be a simple artifact of PH

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