- Email: [email protected]
Dynamin family of mechanoenzymes
454
Dynamin family of mechanoenzymes Dganit Danino* and Jenny E Hinshaw† The dynamin family of proteins is continually growing, and in recent years members have been localized to areas of mitochondrial fission, plant phragmoplasts and chloroplasts, and viral ribonucleoprotein complexes. All the dynamin-like proteins examined to-date appear to assemble into oligomers, such as rings or spirals; however, it remains to be determined if a global mechanism of action exists. Even the role of dynamin in vesicle formation remains controversial as to whether it behaves as a molecular switch or as a mechanochemical enzyme. Addresses Laboratory of Cell Biochemistry and Biology, Building 8, Room 419, MSC 0851, 8 Center Drive, National Institute of Health, Bethesda, Maryland 20892, USA. *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Cell Biology 2001, 13:454–460 0955-0674/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations ADLs Arabidopsis dynamin-like GAP GTPase-activating protein GED GTPase effector domain hGBP1 human guanylate-binding protein 1 LZ leucine zipper PKC protein kinase C PRD proline/arginine-rich domain TGN trans-Golgi network Vps vacuolar protein sorting
Introduction Proteins of the dynamin family are large GTPases implicated in numerous fundamental cellular processes, including several membrane fission events, anti-viral activity, plant cell plate formation and chloroplast biogenesis (Figure 1; for review see [1]). Although they share common structural characteristics, the overall degree of similarity and homology varies among the different family members, in agreement with their diverse functions. Dynamin contains five distinct domains (Figure 2a): a large amino-terminal GTPase domain, containing three GTP-binding motifs and a self-assembly region; a middle domain with potential self-assembly properties; a pleckstrin homology domain involved in membrane binding; a coiled-coil domain (also called a GTPase effector domain, GED) that stimulates the GTPase activity and participates in self-assembly; and a proline/arginine-rich domain (PRD) that was found to increase dynamin–dynamin interactions and contains several SH3-binding sites for binding dynamin partners. Although they all share high sequence homology in the GTPase domain, and they all are believed to have middle and assembly coiled-coil regions, the PH domain is found only in dynamin and ADL3, a plant dynamin-like protein, and only dynamin contains a carboxy-terminal PRD. Other motifs found within dynamin family members, which confer particular functions, are discussed below.
Biochemically, dynamin and dynamin-like proteins have a relatively low affinity for GTP and a high rate of stimulated GTP hydrolysis, which is concentration-dependent. Self-assembly and oligomerization into ordered structures (i.e. rings and spirals) is another common characteristic among these proteins and, for the majority, is essential for their function. Indeed, the three domains involved in selfassembly appear to exist in all dynamin family members. However, for some family members it remains unclear whether monomers or oligomers are the active form. In this review, we focus on the structural and functional similarities within the dynamin family of proteins. While they are continually being implicated in diverse functions of the cell, it is unclear whether they share a common mechanism of action. For dynamin, the data clearly indicate that it is a force-generating molecule capable of constricting an underlying membrane.
Dynamin family members involved in vesicle trafficking Dynamin
In the 1980s the Drosophila homologue of dynamin, shibire, was found to be a major component of endocytosis [2]. Since then dynamin has been implicated in a number of other major cellular processes including receptor-mediated endocytosis, caveolae internalization and membrane trafficking from late endosomes and Golgi [3]. The neuronal isoform of dynamin is phosphorylated by protein kinase C (PKC) [4] and dephosphorylated by calceneurin [5]. Upon depolarization of the synapse, dynamin is dephosphorylated where it is believed to shift from the cytosol to the plasma membrane and localize to coated pits [6]. Upon GTP binding, it redistributes to the necks where it plays a direct role in membrane constriction and possibly membrane fission [1]. Several proteins bind to dynamin through its SH3binding domains and potentially target dynamin to the membrane and/or regulate dynamin’s function. Two of these proteins, amphiphysin and endophilin, colocalize with dynamin to the necks of coated pits [7,8]. Additional molecules such as cell signaling components (i.e. Grb2, PLCγ, Src, PIP2) [9-11] and potential linkers to the cytoskeleton (cortactin) [12•] also bind to dynamin. Purified dynamin exists as a tetramer and self-assembles into rings and spirals [13]. It also assembles on lipid bilayers to form helical tubes [14] that resemble structures seen in nerve termini [2,15]. Upon GTP addition, the dynamin lipid-tubes constrict and fragment, demonstrating that dynamin is a force-generating molecule [14,16]. The GED of dynamin stimulates the GTPase activity (acting as a GAP) and promotes self-assembly by interacting with the GTPase domain [17,18]. GED also forms homodimers or tetramers, and binds to the middle domain [19]. Mutations in this region can lead to an accumulation of constricted coated pits [20••]. Cells
Dynamin family of mechanoenzymes Danino and Hinshaw
455
Figure 1 Dynamin family members are involved in numerous cellular processes. This schematic diagram illustrates the location of dynamin-like proteins within animal and yeast cells (left) and within plant cells (right). Below is a table corresponding to the diagram which indicates the location, function and self-assembly properties of the dynamin family members. Specific proteins are identified by their color in the schematic diagram and table.
Clathrin
Recycling endosome
Mitochondria Cell wall Golgi
Virus
Nucleus
Chloroplast Animal & yeast
Protein
Localization
Plant
Function
Self-assembly
Dynamin
Plasma membrane (clathrin coated, Vesicle formation, caveolae), Golgi,endosomes fission
Vps1
Golgi
+ Unknown
Mgm1/Msp1/OPA1 Mitochondria inner or outer membrane, or matrix
Vesicle formation and transport Mitochondrial fission & morphology Mitochondrial morphology
Phragmoplastin
Cell wall
Membrane morphology
+
ADL1
Cell wall, chloroplast
Membrane biogenesis
+
ADL2
Chloroplast
Unknown
hGBP1
Cytoplasm
Anti-viral activity
+
Mx
Cytoplasm, nucleus
Anti-viral activity
+
Dnm1/Drp1/DRP-1 Mitochondria outer membrane
+ Unknown
Unknown
Current Opinion in Cell Biology
transfected with GED mutants produced conflicting results as to whether dynamin acts as a molecular switch or as a mechanochemical enzyme [18,20••,21•]. Just as dynamin is its own GAP, dynamin may also function as a molecular switch and have mechanochemical properties. Vps1
Yeast Vps proteins (vacuolar protein sorting) control trafficking between the trans-Golgi network (TGN) and endosomes.
Vps1, a dynamin family member, is localized to the Golgi and is involved in clathrin-mediated vesicle formation at the TGN [22], transport of proteins to the vacuole and protein transport from the Golgi to the endosomal system [23]. Vps1 is the only yeast dynamin-like protein known to date that is involved in vesicular transport. It is structurally similar to dynamin but has two inserts, located after the first GTP-binding motif and between the middle and assembly domains, which are also found in several mitochondrial members of the family.
456
Membranes and sorting
Figure 2 (a)
300 GTPase
(b)
521 Middle
623 PH
750 GED
864 PRD
(c) r
Head Stalk a Leg Lipid bilayer
Current Opinion in Cell Biology
Dynamin family members involved in mitochondrial morphology Mitochondrial division is another crucial cellular process that requires membrane fission. It is therefore intriguing that several groups studying different eucaryotic organisms have shown a direct link between dynamin-like proteins and mitochondrial fission. Though outer and inner mitochondrial membrane fission events may be coupled, two separate groups of dynamin-like proteins appear to be involved in these events. Saccharomyces cerevisiae Dnm1, Caenorhabditis elegans DRP-1 and mammalian Drp1 are homologues involved in outer mitochondria membrane fission, whereas the homologues S. cerevisiae Mgm1, Schizosaccharomyces pombe Msp1 and human OPA1 possibly regulate inner membrane mitochondrial fission. Dnm1, Drp1 and DRP-1
Dnm1, Drp1 and DRP-1 are cytoplasmic proteins that assemble on the outer mitochondrial membrane in punctate structures associated with sites of membrane constriction and fission, strongly suggesting that they promote fission of mitochondria [24–29]. However, mutations in Dnm1 that affect mitochondrial division and morphology did not affect mitochondrial DNA inheritance [25–27]. Mutations in the GTPase domain of Drp1 that induce tubule clustering and alter the overall mitochondrial morphology have no effect on the secretory or the endocytic pathway [28]. As Drp1 is localized to the outer mitochondrial membrane, but is also found in the cytosol, it was suggested that it establishes mitochondrial morphology by participating in pinching off mitochondrial fragments and
Structure of the dynamin molecule. (a) A schematic diagram of dynamin’s five domains: GTPase, middle, pleckstrin homology (PH), GTPase effector domain (GED) and prolinerich domain (PRD). Yellow lines in the GTPase domain indicate the position of the three consensus GTP-binding domains and the black lines above the sequence mark the regions involved in self-assembly. (b) and (c) illustrate the 3D reconstruction of ∆PRDdynamin in the constricted state. (b) Surface rendering of the 3D map of ∆PRD-dynamin, showing three prominent density peaks colored green (head), blue (stalk) and gold (leg). In the presence of GMP-PCP, ∆PRDdynamin constricts in the radial (r) and axial (a) directions (arrows). The constricted dynamin-tube is 40 nm in diameter compared to 50 nm for the non-constricted tube. In the axial direction, the tube constricts ~4 nm. (c) A cross-section through the three-dimensional map, illustrating possible interactions within and between dynamin molecules in the dimer. It has been proposed that the GTPase domain of dynamin resides in the head region, the middle and GED reside in the stalk and the PH domain resides in the leg. The leg region inserts into the outer leaflet of the lipid bilayer.
distributing them throughout the cytoplasm, analogous to dynamin’s role in endocytosis [28,30]. Drp1, also referred to in the literature as DVLP [31,32], DLP1 [33,34], Dymple [35,36] and human dynamin IV (HdynIV) [37], assembles into large aggregates at low salt concentrations and possibly exists as a tetramer under physiological conditions [32], similar to dynamin [13]. Interdomain interactions between regions along the molecule are similar to those found in dynamin, suggesting a similar assembly mechanism [32]. Mgm1, Msp1 and OPA1
Mgm1, Msp1 and OPA1 have a long, basic-rich aminoterminal extension that is required for mitochondrial localization. Mgm1 is mainly implicated in the determination of normal mitochondrial morphology and division. Its role in maintenance of the mitochondria genome [38] appears to be a secondary effect to the morphological changes [39]. The exact localization of Mgm1 remains controversial. Shepard and Yaffe [39] have shown that Mgm1 associates with the mitochondrial outer membrane, possibly via a domain in the amino terminus. However, recently Wong et al. [40••] have shown that Mgm1 is localized to the intermembrane space and possibly functions in inner membrane morphological events such as inner membrane fission, in analogy to dynamin. Msp1, the homologue of Mgm1 in S. pombe [41], is possibly essential for maintenance of mitochondrial DNA [42]. Msp1 is anchored to the matrix side of the mitochondrial inner membrane through two transmembrane segments in
Dynamin family of mechanoenzymes Danino and Hinshaw
its amino terminus [42]. The first amino-terminal residues interact with a cell cycle regulator in yeast, suggesting a connection between mitochondrial biogenesis and function, and cell cycle machinery [42,43]. OPA1, the human homologue, is the only known human dynamin-like protein that has the amino-terminal mitochondria leader. This protein is widely expressed and most abundant in the retina [44,45]. Mutations, including a mutation in the GTPase domain, are related to optic atrophy type 1 (OPA1), an inherited optical neuropathy disease [44]. The authors suggest that modification in OPA1 may cause impairment of the integrity and/or function of mitochondria, similar to that found in yeast.
Plant dynamin family members Phragmoplastin and ADLs (Arabidopsis dynamin-like) are higher plant members of the dynamin-like family. Phragmoplastin and ADL1 are membrane-associated proteins that accumulate at the plane of cell wall formation during cytokinesis [46–48], a process by which a new plasma membrane and cell wall form by fusion of Golgi-derived vesicles in the plane of cell division. Their exact function in this process is not known, yet they are likely to be involved in membrane remodeling events. Homo- and hetero-oligomers of phragmoplastin form through interactions between an assembly region located at the GTP-binding domain and a self-assembly domain in the middle of the molecule [49•]. At physiological conditions, purified phragmoplastin exists predominantly as monomers and dimers but when dialyzed into low salt, higher-order helical assemblies form [49•], as was found with dynamin [50]. GTP induces disassembly, whereas GTPγS makes the helical structures more compact, a conformation change that is also observed with ∆PRD dynamin (P Zhang, JE Hinshaw, unpublished results). ADLs are present in multiple forms in Arabidopsis leaf tissue, partially as high molecular complexes [51]. ADL1 is localized to thylakoid membranes of chloroplasts, possibly on the outward-facing side, and it is probably involved in thylakoid membrane biogenesis and vesicle formation [52]. ADL2, which also localizes to chloroplasts, is most closely related to Dnm1 of yeast [53]. Surprisingly, ADL3 has a PH domain [54], located between the middle and coiled-coil domains, similar to the location of this region in dynamin. ADL3 possibly associates with membranes through interactions between its PH domain and membranes phosphoiniositides [54]. Although the location of ADL3 is unclear, its domain similarity to dynamin suggests that it is involved in similar cellular functions.
Interferon-induced dynamin family members Human guanylate-binding protein 1 (hGBP1) and Mx are dynamin family proteins found to be among the most abundant proteins induced by interferon-γ. Despite their abundance, their biological function is poorly understood however, the study of hGBP1 and Mx is an exciting and growing field.
457
Human guanylate-binding protein 1
hGBP1 blocks the replication of several viruses [55]. The mechanism of this antiviral activity has not been established, but it may be coupled to nucleotide binding or GTP hydrolysis [56] as with the Mx proteins [57]. hGBP1 binds GTP, GDP and GMP with equal affinity [56], hydrolyzes GTP into both GDP and GMP [58,59] and oligomerizes in a nucleotide-binding-dependent manner. While it is a monomer in the absence of nucleotide and in complex with GMP or GDP, it forms dimers in the presence of GTP or a non-hydrolyzable GTP analogue, GppNHp, and possibly tetramers in complex with GDP and aluminum fluoride. As with dynamin, oligomerization may be required for efficient GTP hydrolysis. Self-assembly of hGBP1 may also play a role in interferon-mediated cellular functions [60••]. The full-length crystal structure of hGBP1 was determined in a nucleotide-free state [60••] and in complex with GppNHp [61]. The models exhibit many similarities to small Ras-related proteins but also several specific differences. A major structural difference is that the phosphate cap of the hGBP1–GppNHp complex (corresponding to the switch I region) shields the phosphates in the binding site from a potential GTPase-activating protein. In addition, the guanine base-site that is open to the solvent in Ras is mostly covered by an insert forming a hydrophobic pocket for the guanine base in hGBP1. It was also shown that the carboxyl terminus interacts with the amino terminus, as is the case for other members of the dynamin family. Mx proteins
Mx proteins are interferon-induced proteins found in vertebrates that exhibit strong anti-viral activity. MxA and MxB are expressed in humans, whereas Mx1, Mx2 and Mx3 are found in rodents. MxA accumulates in the cytoplasm in response to type I (α/β) interferon and inhibits multiplication of several types of RNA viruses. It forms homo-oligomers through a central interactive region and a conserved leucine zipper (LZ) element. The latter motif is responsible for intra- and intermolecular interactions and regulates both GTPase activity and viral target recognition [62,63]. Recently, it was shown that MxA oligomers interact with viral ribonucleoprotein complexes (vRNPs). This interaction retains the virus in the cytoplasm and prevents the translocation of its genome into the nucleus [64,65]. This process depends on the presence of GTPγS as a stabilizing factor [63,66], indicating that GTP binding, but not hydrolysis, is required for the antiviral activity. Apparently, GTP binding induces a conformational change that allows tight binding of the viral structures [67•]. Anti-viral activity was also detected in the presence of MxA mutants that failed to assemble and could not hydrolyze GTP in vivo, suggesting for the first time that monomers may be the active form of a dynamin-like protein [67•]. Cytoplasmic and nuclear isoforms of MxB are typically found in a granular pattern [68]. Nuclear MxB associates with heterochromatin beneath the nuclear envelope and is targeted to the nucleus through a hydrophobic ‘functional nuclear
458
Membranes and sorting
localization’ element located at the first amino acids of the amino terminus. It forms hetero-oligomers with cytoplasmic MxB through the LZ element, enabling it to enter the nucleus [69]. MxA and MxB do not form hetero-oligomers, probably due to differences in the leucine zipper sequence of the two proteins. Surprisingly, no anti-viral activity has been found for human MxB protein, and its function is still unclear. However, all the Mx proteins probably have a normal cellular function that has yet to be determined.
Conclusions Members of the dynamin family consist of a diverse group of proteins with numerous cellular locations and functions. As more members are identified, several distinct subgroups may emerge that are involved in general phenomena such as membrane fission events or anti-viral activity. A recently discovered dynamin-like protein, mG120, may represent a new class of dynamin-like proteins directly associated with the cytoskeleton. mG120, the most divergent member, contains a carboxy-terminal region that is homologous to the myosin heavy chain [70]. A common property of all the proteins of the dynamin family may be to self-assemble into higher ordered oligomers on specific templates (i.e. membranes and vRNPs). Another common property of the dynamin-like proteins may be their ability to undergo a conformational change upon GTP binding or hydrolysis, which then modifies their underlying substrate. The precise mechanism of action for these proteins is currently being pursued and the study of this family of large GTPases over the next few years promises to be very exciting and informative.
Update The first three-dimensional structure of dynamin has recently been solved using high-resolution cryo-electron microscopy (P Zhang, JE Hinshaw, unpublished data) (Figure 2b,c). The three-dimensional map of a dynamin mutant missing the PRD (∆PRD) was determined in the constricted state (+GMPPCP) and revealed a ‘T’ shaped dimer consisting of three prominent densities: leg, stalk and head (Figure 2b,c). The structure suggests that the dense stalk and head groups undergo a global conformational change upon GTP binding or hydrolysis that generates a force on the underlying lipid bilayer leading to membrane constriction. These results clearly suggest that regardless of whether dynamin acts as a molecular switch, targeting other fission components to the neck region, dynamin is clearly a force generating ‘constrictase’.
Acknowledgements We wish to thank JM Shaw for her excellent comments on the manuscript. We thank B Danino and N Dwyer for excellent technical assistance.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Hinshaw JE: Dynamin and its role in membrane fission. Annu Rev Cell Dev Biol 2000, 16:483-519.
2.
Kosaka T, Ikeda K: Possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in Drosophila. J Neurobiol 1983, 14:207-225.
3.
Schmid SL, McNiven MA, De Camilli P: Dynamin and its partners: a progress report. Curr Opin Cell Biol 1998, 10:504-512.
4.
Robinson PJ, Sontag JM, Liu JP, Fykse EM, Slaughter C, McMahon H, Sudhof TC: Dynamin GTPase regulated by protein kinase C phosphorylation in nerve terminals. Nature 1993, 365:163-166.
5.
Liu JP, Sim ATR, Robinson PJ: Calcineurin inhibition of dynamin I GTPase activity coupled to nerve terminal depolarization. Science 1994, 265:970-972.
6.
Liu JP, Powell KA, Sudhof TC, Robinson PJ: Dynamin I is a Ca2++sensitive phospholipid-binding protein with very high affinity for protein kinase C•. J Biol Chem 1994, 269:21043-21050.
7.
Takei K, Slepnev VI, Haucke V, De Camilli P: Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nat Cell Biol 1999, 1:33-39.
8.
Ringstad N, Gad H, Low P, Di Paolo G, Brodin L, Shupliakov O, De Camilli P: Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis. Neuron 1999, 24:143-154.
9.
Seedorf K, Kostka G, Lammers R, Bashkin P, Daly R, Burgess WH, van der Bliek AM, Schlessinger J, Ullrich A: Dynamin binds to SH3 domains of phospholipase C gamma and GRB-2. J Biol Chem 1994, 269:16009-16014.
10. Salim K, Bottomley MJ, Querfurth E, Zvelebil MJ, Gout I, Scaife R, Margolis RL, Gigg R, Smith CI, Driscoll PC et al.: Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton’s tyrosine kinase. EMBO J 1996, 15:6241-6250. 11. Gout I, Dhand R, Hiles ID, Fry MJ, Panayotou G, Das P, Truong O, Totty NF, Hsuan J, Booker GW: The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell 1993, 75:25-36. 12. McNiven MA, Kim L, Krueger EW, Orth JD, Cao H, Wong TW: • Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape. J Cell Biol 2000, 151:187-198. In this manuscript, dynamin 2 was localized to membrane ruffles and lamellipodia and the evidence strongly suggests dynamin interaction with cortactin in these regions. This demonstrates a direct link between dynmain and the actin cytoskeleton. 13. Hinshaw JE: Dynamin spirals. Curr Opin Struct Biol 1999, 9:260-267. 14. Sweitzer SM, Hinshaw JE: Dynamin undergoes a GTP-dependent conformational change causing vesiculation. Cell 1998, 93:1021-1029. 15. Takei K, McPherson PS, Schmid SL, De Camilli P: Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals. Nature 1995, 374:186-190. 16. Takei K, Haucke V, Slepnev V, Farsad K, Salzar M, Chen H, De Camilli P: Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes. Cell 1998, 94:131-141. 17.
Muhlberg AB, Warnock DE, Schmid SL: Domain structure and intramolecular regulation of dynamin GTPase. EMBO J 1997, 16:6676-6683.
18. Sever S, Muhlberg AB, Schmid SL: Impairment of dynamin’s GAP domain stimulates receptor-mediated endocytosis. Nature 1999, 398:481-486. 19. Smirnova E, Shurland DL, Newman-Smith ED, Pishvaee B, van der Bliek AM: A model for dynamin self-assembly based on binding between three different protein domains. J Biol Chem 1999, 274:14942-14947. 20. Sever S, Damke H, Schmid SL: Dynamin: GTP controls the •• formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis. J Cell Biol 2000, 150:1137-1148. The authors have further characterized 2 dynamin mutants in the GED region in vivo. One mutant is defective in stimulated GTPase activity and was shown to accumulate constricted coated pits. The other mutant, defective in self-assembly, increased rates of vesicle formation. From these studies and previous work, the authors suggest that dynamin acts as a classical GTPase.
Dynamin family of mechanoenzymes Danino and Hinshaw
21. Marks B, Stowell MHB, Vallis Y, Mills IG, Gibson A, Hopkins CR, • McMahon HT: GTPase activity of dynamin and resulting conformation change are essential for endocytosis. Nature 2001, 410:231-235. Using the same mutants described in [18,20••], the authors suggest in this manuscript that the GED mutants have normal stimulated GTPase activity and do not affect endocytosis. These conflicting results may be due to the different systems used to examine the mutants. 22. Bensen ES, Costaguta G, Payne GS: Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae. Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network. Genetics 2000, 154:83-97.
459
40. Wong ED, Wagner JA, Gorsich SW, McCaffery JM, Shaw JM, •• Nunnari J: The dynamin-related GTPase, Mgm1p, is an intermembrane space protein required for maintenance of fusion competent mitochondria. J Cell Biol 2000, 151:341-352. In this manuscript, the authors were surprised to observe an increase in mitochondrial fission in MGM1-deleted cells. They suggest that Mgm1p is not directly involved in fusion, instead the deletion of Mgm1p causes an uncoupling between the outer and inner mitochondrial machinery, increasing the outer mitochondrial fission events. 41. Pelloquin L, Belenguer P, Menon Y, Ducommun B: Identification of a fission yeast dynamin-related protein involved in mitochondrial DNA maintenance. Biochem Biophys Res Commun 1998, 251:720-726.
23. Luo WJ, Chang A: An endosome-to-plasma membrane pathway involved in trafficking of a mutant plasma membrane ATPase in yeast. Mol Biol Cell 2000, 11:579-592.
42. Pelloquin L, Belenguer P, Menon Y, Gas N, Ducommun B: Fission yeast Msp1 is a mitochondrial dynamin-related protein. J Cell Sci 1999, 112:4151-4161.
24. Fekkes P, Shepard KA, Yaffe MP: Gag3p, an outer membrane protein required for fission of mitochondrial tubules. J Cell Biol 2000, 151:333-340.
43. Pelloquin L, Ducommun B, Belenguer P: Interaction between the fission yeast nim1/cdr1 protein kinase and a dynamin-related protein. FEBS Lett 1999, 443:71-74.
25. Bleazard W, McCaffery JM, King EJ, Bale S, Mozdy A, Tieu Q, Nunnari J, Shaw JM: The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat Cell Biol 1999, 1:298-304.
44. Alexander C, Votruba M, Pesch UE, Thiselton DL, Mayer S, Moore A, Rodriguez M, Kellner U, Leo-Kottler B, Auburger G et al.: OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nat Genet 2000, 26:211-215.
26. Sesaki H, Jensen RE: Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape. J Cell Biol 1999, 147:699-706. 27.
Otsuga D, Keegan BR, Brisch E, Thatcher JW, Hermann GJ, Bleazard W, Shaw JM: The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast. J Cell Biol 1998, 143:333-349.
28. Smirnova E, Shurland DL, Ryazantsev SN, van der Bliek AM: A human dynamin-related protein controls the distribution of mitochondria. J Cell Biol 1998, 143:351-358. 29. Labrousse AM, Shurland DL, van der Bliek AM: Contribution of the GTPase domain to the subcellular localization of dynamin in the nematode Caenorhabditis elegans. Mol Biol Cell 1998, 9:3227-3239. 30. van der Bliek AM: A mitochondrial division apparatus takes shape. J Cell Biol 2000, 151:F1-F4. 31. Shin HW, Shinotsuka C, Torii S, Murakami K, Nakayama K: Identification and subcellular localization of a novel mammalian dynamin-related protein homologous to yeast Vps1p and Dnm1p. J Biochem (Tokyo) 1997, 122:525-30. 32. Shin HW, Takatsu H, Mukai H, Munekata E, Murakami K, Nakayama K: Intermolecular and interdomain interactions of a dynamin-related GTP-binding protein, Dnm1p/Vps1p-like protein. J Biol Chem 1999, 274:2780-2785. 33. Yoon YS, Pitts KR, Oswald BJ, McNiven MA: The dynamin-like protein (DLP1) induces membrane tubulation in living cells. Mol Biol Cell 1999, 10:314A-314A. 34. Imoto M, Tachibana I, Urrutia R: Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps1p. J Cell Sci 1998, 111:1341-1349. 35. Kamimoto T, Nagai Y, Onogi H, Muro Y, Wakabayashi T, Hagiwara M: Dymple, a novel dynamin-like high molecular weight GTPase lacking a proline-rich carboxyl-terminal domain in mammalian cells. J Biol Chem 1998, 273:1044-1051. 36. Muro Y, Kamimoto T, Tomita Y, Hagiwara M: Spectrum of autoantibodies against a dynamin-related protein, dymple. Arthritis Rheum 2000, 43:1516-1519. 37.
Hong YR, Chen CH, Cheng DS, Howng SL, Chow CC: Human dynamin-like protein interacts with the glycogen synthase kinase β. Biochem Biophys Res Commun 1998, 249:697-703. 3β
38. Jones BA, Fangman WL: Mitochondrial DNA maintenance in yeast requires a protein containing a region related to the GTP-binding domain of dynamin. Genes Dev 1992, 6:380-389. 39. Shepard KA, Yaffe MP: The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance. J Cell Biol 1999, 144:711-720.
45. Delettre C, Lenaers G, Griffoin JM, Gigarel N, Lorenzo C, Belenguer P, Pelloquin L, Grosgeorge J, Turc-Carel C, Perret E et al.: Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nat Genet 2000, 26:207-210. 46. Gu X, Verma DPS: Phragmoplastin, a dynamin-like protein associated with cell plate formation in plants. EMBO J 1996, 15:695-704. 47.
Gu X, Verma DPS: Dynamics of phragmoplastin in living cells during cell plate formation and uncoupling of cell elongation from the plane of cell division. Plant Cell 1997, 9:157-169.
48. Waizenegger I, Lukowitz W, Assaad F, Schwarz H, Jurgens G, Mayer U: The Arabidopsis KNOLLE and KEULE genes interact to promote vesicle fusion during cytokinesis. Curr Biol 2000, 10:1371-1374. 49. Zhang Z, Hong Z, Verma DPS: Phragmoplastin polymerizes into • spiral coiled structures via intermolecular interaction of two selfassembly domains. J Biol Chem 2000, 275:8779-8784. Two self-assembly domains were identified in phragmoplastin, and phragmoplastin was shown to self-assemble into higher ordered structures in low salt or upon addition of GTP-gamma-S. This further demonstrates that selfassembly is a common property among the dynamin-like proteins. 50. Hinshaw JE, Schmid SL: Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature 1995, 374:190-192. 51. Park JM, Kang SG, Pih KT, Jang HJ, Piao HL, Yoon HW, Cho MJ, Hwang I: A dynamin-like protein, ADL1, is present in membranes as a high-molecular-mass complex in Arabidopsis thaliana. Plant Physiol 1997, 115:763-771. 52. Park JM, Cho JH, Kang SG, Jang HJ, Pih KT, Piao HL, Cho MJ, Hwang I: A dynamin-like protein in Arabidopsis thaliana is involved in biogenesis of thylakoid membranes. EMBO J 1998, 17:859-867. 53. Kang SG, Jin JB, Piao HL, Jang HJ, Lim JH, Hwang I: Molecular cloning of an Arabidopsis cDNA encoding a dynamin-like protein that is localized to plastids. Plant Mol Biol 1998, 38:437-447. 54. Mikami K, Iuchi S, Yamaguchi-Shinozaki K, Shinozaki K: A novel Arabidopsis thaliana dynamin-like protein containing the pleckstrin homology domain. J Exp Bot 2000, 51:317-318. 55. Anderson SL, Carton JM, Lou J, Xing L, Rubin BY: Interferoninduced guanylate binding protein-1 (GBP-1) mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus. Virology 1999, 256:8-14. 56. Praefcke GJ, Geyer M, Schwemmle M, Robert Kalbitzer H, Herrmann C: Nucleotide-binding characteristics of human guanylate-binding protein 1 (hGBP1) and identification of the third GTP-binding motif. J Mol Biol 1999, 292:321-332.
460
57.
Membranes and sorting
Schwemmle M, Richter MF, Hermann C, Nassar N, Staeheli P: Unexpected structural requirements for GTPase activity of the interferon-induced MxA protein. J Biol Chem 1995, 270:13518-13523.
65. Weber F, Haller O, Kochs G: MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes. J Virol 2000, 74:560-563.
58. Schwemmle M, Staeheli P: The interferon-induced 67-kDa guanylate-binding protein (hGBP1) is a GTPase that converts GTP to GMP. J Biol Chem 1994, 269:11299-11305.
66. Kochs G, Haller O: GTP-bound human MxA protein interacts with the nucleocapsids of Thogoto virus (Orthomyxoviridae). J Biol Chem 1999, 274:4370-4376.
59. Neun R, Richter MF, Staeheli P, Schwemmle M: GTPase properties of the interferon-induced human guanylate-binding protein 2. FEBS Lett 1996, 390:69-72.
67. Janzen C, Kochs G, Haller O: A monomeric GTPase-negative MxA • mutant with antiviral activity. J Virol 2000, 74:8202-8206. An MxA mutant, lacking GTPase activity, was shown to retain anti-viral activity. This mutant is predicted to be defective in oligomerization and therefore suggests MxA is active in the monomeric state. This is the first dynamin-like protein predicted to be active in the monomeric form.
60. Prakash B, Praefcke GJ, Renault L, Wittinghofer A, Herrmann C: •• Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins. Nature 2000, 403:567-571. This manuscript describes the first X-ray crystal structure of a dynamin-like protein. The structure reveals a large globular GTP-binding domain with an elongated alpha-helical domain that loops back to the GTP-binding domain. 61. Prakash B, Renault L, Praefcke GJ, Herrmann C, Wittinghofer A: Triphosphate structure of guanylate-binding protein 1 and implications for nucleotide binding and GTPase mechanism. EMBO J 2000, 19:4555-4564. 62. Flohr F, SSS, Haller O, Kochs G: The central interactive region of human MxA GTPase is involved in GTPase activation and interaction with viral target structures. FEBS Lett 1999, 463:24-28. 63. Kochs G, Trost M, Janzen C, Haller O: MxA GTPase: oligomerization and GTP-dependent interaction with viral RNP target structures. Methods 1998, 15:255-263. 64. Kochs G, Haller O: Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids. Proc Natl Acad Sci USA 1999, 96:2082-2086.
68. Melen K, Keskinen P, Ronni T, Sareneva T, Lounatmaa K, Julkunen I: Human MxB protein, an interferon-alpha-inducible GTPase, contains a nuclear targeting signal and is localized in the heterochromatin region beneath the nuclear envelope. J Biol Chem 1996, 271:23478-23486. 69. Melen K, Julkunen I: Nuclear cotransport mechanism of cytoplasmic human MxB protein. J Biol Chem 1997, 272:32353-32359. 70. Kubokawa K, Miyashita T, Kubo Y: Isolation of a cDNA for a novelkDa GTP-binding protein expressed in motor neurons in the salmon brain. FEBS Lett 1998, 431:231-235.
Note added in proof The work referred to in the text as (P Zhang, JE Hinshaw, unpublished data) is now in press: 71. Zhang P, Hinshaw JE: Three dimensional reconstruction of dynamin in the constricted state. Nat Cell Biol 2001, in press.
Dynamin family of mechanoenzymes Dganit Danino* and Jenny E Hinshaw† The dynamin family of proteins is continually growing, and in recent years members have been localized to areas of mitochondrial fission, plant phragmoplasts and chloroplasts, and viral ribonucleoprotein complexes. All the dynamin-like proteins examined to-date appear to assemble into oligomers, such as rings or spirals; however, it remains to be determined if a global mechanism of action exists. Even the role of dynamin in vesicle formation remains controversial as to whether it behaves as a molecular switch or as a mechanochemical enzyme. Addresses Laboratory of Cell Biochemistry and Biology, Building 8, Room 419, MSC 0851, 8 Center Drive, National Institute of Health, Bethesda, Maryland 20892, USA. *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Cell Biology 2001, 13:454–460 0955-0674/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations ADLs Arabidopsis dynamin-like GAP GTPase-activating protein GED GTPase effector domain hGBP1 human guanylate-binding protein 1 LZ leucine zipper PKC protein kinase C PRD proline/arginine-rich domain TGN trans-Golgi network Vps vacuolar protein sorting
Introduction Proteins of the dynamin family are large GTPases implicated in numerous fundamental cellular processes, including several membrane fission events, anti-viral activity, plant cell plate formation and chloroplast biogenesis (Figure 1; for review see [1]). Although they share common structural characteristics, the overall degree of similarity and homology varies among the different family members, in agreement with their diverse functions. Dynamin contains five distinct domains (Figure 2a): a large amino-terminal GTPase domain, containing three GTP-binding motifs and a self-assembly region; a middle domain with potential self-assembly properties; a pleckstrin homology domain involved in membrane binding; a coiled-coil domain (also called a GTPase effector domain, GED) that stimulates the GTPase activity and participates in self-assembly; and a proline/arginine-rich domain (PRD) that was found to increase dynamin–dynamin interactions and contains several SH3-binding sites for binding dynamin partners. Although they all share high sequence homology in the GTPase domain, and they all are believed to have middle and assembly coiled-coil regions, the PH domain is found only in dynamin and ADL3, a plant dynamin-like protein, and only dynamin contains a carboxy-terminal PRD. Other motifs found within dynamin family members, which confer particular functions, are discussed below.
Biochemically, dynamin and dynamin-like proteins have a relatively low affinity for GTP and a high rate of stimulated GTP hydrolysis, which is concentration-dependent. Self-assembly and oligomerization into ordered structures (i.e. rings and spirals) is another common characteristic among these proteins and, for the majority, is essential for their function. Indeed, the three domains involved in selfassembly appear to exist in all dynamin family members. However, for some family members it remains unclear whether monomers or oligomers are the active form. In this review, we focus on the structural and functional similarities within the dynamin family of proteins. While they are continually being implicated in diverse functions of the cell, it is unclear whether they share a common mechanism of action. For dynamin, the data clearly indicate that it is a force-generating molecule capable of constricting an underlying membrane.
Dynamin family members involved in vesicle trafficking Dynamin
In the 1980s the Drosophila homologue of dynamin, shibire, was found to be a major component of endocytosis [2]. Since then dynamin has been implicated in a number of other major cellular processes including receptor-mediated endocytosis, caveolae internalization and membrane trafficking from late endosomes and Golgi [3]. The neuronal isoform of dynamin is phosphorylated by protein kinase C (PKC) [4] and dephosphorylated by calceneurin [5]. Upon depolarization of the synapse, dynamin is dephosphorylated where it is believed to shift from the cytosol to the plasma membrane and localize to coated pits [6]. Upon GTP binding, it redistributes to the necks where it plays a direct role in membrane constriction and possibly membrane fission [1]. Several proteins bind to dynamin through its SH3binding domains and potentially target dynamin to the membrane and/or regulate dynamin’s function. Two of these proteins, amphiphysin and endophilin, colocalize with dynamin to the necks of coated pits [7,8]. Additional molecules such as cell signaling components (i.e. Grb2, PLCγ, Src, PIP2) [9-11] and potential linkers to the cytoskeleton (cortactin) [12•] also bind to dynamin. Purified dynamin exists as a tetramer and self-assembles into rings and spirals [13]. It also assembles on lipid bilayers to form helical tubes [14] that resemble structures seen in nerve termini [2,15]. Upon GTP addition, the dynamin lipid-tubes constrict and fragment, demonstrating that dynamin is a force-generating molecule [14,16]. The GED of dynamin stimulates the GTPase activity (acting as a GAP) and promotes self-assembly by interacting with the GTPase domain [17,18]. GED also forms homodimers or tetramers, and binds to the middle domain [19]. Mutations in this region can lead to an accumulation of constricted coated pits [20••]. Cells
Dynamin family of mechanoenzymes Danino and Hinshaw
455
Figure 1 Dynamin family members are involved in numerous cellular processes. This schematic diagram illustrates the location of dynamin-like proteins within animal and yeast cells (left) and within plant cells (right). Below is a table corresponding to the diagram which indicates the location, function and self-assembly properties of the dynamin family members. Specific proteins are identified by their color in the schematic diagram and table.
Clathrin
Recycling endosome
Mitochondria Cell wall Golgi
Virus
Nucleus
Chloroplast Animal & yeast
Protein
Localization
Plant
Function
Self-assembly
Dynamin
Plasma membrane (clathrin coated, Vesicle formation, caveolae), Golgi,endosomes fission
Vps1
Golgi
+ Unknown
Mgm1/Msp1/OPA1 Mitochondria inner or outer membrane, or matrix
Vesicle formation and transport Mitochondrial fission & morphology Mitochondrial morphology
Phragmoplastin
Cell wall
Membrane morphology
+
ADL1
Cell wall, chloroplast
Membrane biogenesis
+
ADL2
Chloroplast
Unknown
hGBP1
Cytoplasm
Anti-viral activity
+
Mx
Cytoplasm, nucleus
Anti-viral activity
+
Dnm1/Drp1/DRP-1 Mitochondria outer membrane
+ Unknown
Unknown
Current Opinion in Cell Biology
transfected with GED mutants produced conflicting results as to whether dynamin acts as a molecular switch or as a mechanochemical enzyme [18,20••,21•]. Just as dynamin is its own GAP, dynamin may also function as a molecular switch and have mechanochemical properties. Vps1
Yeast Vps proteins (vacuolar protein sorting) control trafficking between the trans-Golgi network (TGN) and endosomes.
Vps1, a dynamin family member, is localized to the Golgi and is involved in clathrin-mediated vesicle formation at the TGN [22], transport of proteins to the vacuole and protein transport from the Golgi to the endosomal system [23]. Vps1 is the only yeast dynamin-like protein known to date that is involved in vesicular transport. It is structurally similar to dynamin but has two inserts, located after the first GTP-binding motif and between the middle and assembly domains, which are also found in several mitochondrial members of the family.
456
Membranes and sorting
Figure 2 (a)
300 GTPase
(b)
521 Middle
623 PH
750 GED
864 PRD
(c) r
Head Stalk a Leg Lipid bilayer
Current Opinion in Cell Biology
Dynamin family members involved in mitochondrial morphology Mitochondrial division is another crucial cellular process that requires membrane fission. It is therefore intriguing that several groups studying different eucaryotic organisms have shown a direct link between dynamin-like proteins and mitochondrial fission. Though outer and inner mitochondrial membrane fission events may be coupled, two separate groups of dynamin-like proteins appear to be involved in these events. Saccharomyces cerevisiae Dnm1, Caenorhabditis elegans DRP-1 and mammalian Drp1 are homologues involved in outer mitochondria membrane fission, whereas the homologues S. cerevisiae Mgm1, Schizosaccharomyces pombe Msp1 and human OPA1 possibly regulate inner membrane mitochondrial fission. Dnm1, Drp1 and DRP-1
Dnm1, Drp1 and DRP-1 are cytoplasmic proteins that assemble on the outer mitochondrial membrane in punctate structures associated with sites of membrane constriction and fission, strongly suggesting that they promote fission of mitochondria [24–29]. However, mutations in Dnm1 that affect mitochondrial division and morphology did not affect mitochondrial DNA inheritance [25–27]. Mutations in the GTPase domain of Drp1 that induce tubule clustering and alter the overall mitochondrial morphology have no effect on the secretory or the endocytic pathway [28]. As Drp1 is localized to the outer mitochondrial membrane, but is also found in the cytosol, it was suggested that it establishes mitochondrial morphology by participating in pinching off mitochondrial fragments and
Structure of the dynamin molecule. (a) A schematic diagram of dynamin’s five domains: GTPase, middle, pleckstrin homology (PH), GTPase effector domain (GED) and prolinerich domain (PRD). Yellow lines in the GTPase domain indicate the position of the three consensus GTP-binding domains and the black lines above the sequence mark the regions involved in self-assembly. (b) and (c) illustrate the 3D reconstruction of ∆PRDdynamin in the constricted state. (b) Surface rendering of the 3D map of ∆PRD-dynamin, showing three prominent density peaks colored green (head), blue (stalk) and gold (leg). In the presence of GMP-PCP, ∆PRDdynamin constricts in the radial (r) and axial (a) directions (arrows). The constricted dynamin-tube is 40 nm in diameter compared to 50 nm for the non-constricted tube. In the axial direction, the tube constricts ~4 nm. (c) A cross-section through the three-dimensional map, illustrating possible interactions within and between dynamin molecules in the dimer. It has been proposed that the GTPase domain of dynamin resides in the head region, the middle and GED reside in the stalk and the PH domain resides in the leg. The leg region inserts into the outer leaflet of the lipid bilayer.
distributing them throughout the cytoplasm, analogous to dynamin’s role in endocytosis [28,30]. Drp1, also referred to in the literature as DVLP [31,32], DLP1 [33,34], Dymple [35,36] and human dynamin IV (HdynIV) [37], assembles into large aggregates at low salt concentrations and possibly exists as a tetramer under physiological conditions [32], similar to dynamin [13]. Interdomain interactions between regions along the molecule are similar to those found in dynamin, suggesting a similar assembly mechanism [32]. Mgm1, Msp1 and OPA1
Mgm1, Msp1 and OPA1 have a long, basic-rich aminoterminal extension that is required for mitochondrial localization. Mgm1 is mainly implicated in the determination of normal mitochondrial morphology and division. Its role in maintenance of the mitochondria genome [38] appears to be a secondary effect to the morphological changes [39]. The exact localization of Mgm1 remains controversial. Shepard and Yaffe [39] have shown that Mgm1 associates with the mitochondrial outer membrane, possibly via a domain in the amino terminus. However, recently Wong et al. [40••] have shown that Mgm1 is localized to the intermembrane space and possibly functions in inner membrane morphological events such as inner membrane fission, in analogy to dynamin. Msp1, the homologue of Mgm1 in S. pombe [41], is possibly essential for maintenance of mitochondrial DNA [42]. Msp1 is anchored to the matrix side of the mitochondrial inner membrane through two transmembrane segments in
Dynamin family of mechanoenzymes Danino and Hinshaw
its amino terminus [42]. The first amino-terminal residues interact with a cell cycle regulator in yeast, suggesting a connection between mitochondrial biogenesis and function, and cell cycle machinery [42,43]. OPA1, the human homologue, is the only known human dynamin-like protein that has the amino-terminal mitochondria leader. This protein is widely expressed and most abundant in the retina [44,45]. Mutations, including a mutation in the GTPase domain, are related to optic atrophy type 1 (OPA1), an inherited optical neuropathy disease [44]. The authors suggest that modification in OPA1 may cause impairment of the integrity and/or function of mitochondria, similar to that found in yeast.
Plant dynamin family members Phragmoplastin and ADLs (Arabidopsis dynamin-like) are higher plant members of the dynamin-like family. Phragmoplastin and ADL1 are membrane-associated proteins that accumulate at the plane of cell wall formation during cytokinesis [46–48], a process by which a new plasma membrane and cell wall form by fusion of Golgi-derived vesicles in the plane of cell division. Their exact function in this process is not known, yet they are likely to be involved in membrane remodeling events. Homo- and hetero-oligomers of phragmoplastin form through interactions between an assembly region located at the GTP-binding domain and a self-assembly domain in the middle of the molecule [49•]. At physiological conditions, purified phragmoplastin exists predominantly as monomers and dimers but when dialyzed into low salt, higher-order helical assemblies form [49•], as was found with dynamin [50]. GTP induces disassembly, whereas GTPγS makes the helical structures more compact, a conformation change that is also observed with ∆PRD dynamin (P Zhang, JE Hinshaw, unpublished results). ADLs are present in multiple forms in Arabidopsis leaf tissue, partially as high molecular complexes [51]. ADL1 is localized to thylakoid membranes of chloroplasts, possibly on the outward-facing side, and it is probably involved in thylakoid membrane biogenesis and vesicle formation [52]. ADL2, which also localizes to chloroplasts, is most closely related to Dnm1 of yeast [53]. Surprisingly, ADL3 has a PH domain [54], located between the middle and coiled-coil domains, similar to the location of this region in dynamin. ADL3 possibly associates with membranes through interactions between its PH domain and membranes phosphoiniositides [54]. Although the location of ADL3 is unclear, its domain similarity to dynamin suggests that it is involved in similar cellular functions.
Interferon-induced dynamin family members Human guanylate-binding protein 1 (hGBP1) and Mx are dynamin family proteins found to be among the most abundant proteins induced by interferon-γ. Despite their abundance, their biological function is poorly understood however, the study of hGBP1 and Mx is an exciting and growing field.
457
Human guanylate-binding protein 1
hGBP1 blocks the replication of several viruses [55]. The mechanism of this antiviral activity has not been established, but it may be coupled to nucleotide binding or GTP hydrolysis [56] as with the Mx proteins [57]. hGBP1 binds GTP, GDP and GMP with equal affinity [56], hydrolyzes GTP into both GDP and GMP [58,59] and oligomerizes in a nucleotide-binding-dependent manner. While it is a monomer in the absence of nucleotide and in complex with GMP or GDP, it forms dimers in the presence of GTP or a non-hydrolyzable GTP analogue, GppNHp, and possibly tetramers in complex with GDP and aluminum fluoride. As with dynamin, oligomerization may be required for efficient GTP hydrolysis. Self-assembly of hGBP1 may also play a role in interferon-mediated cellular functions [60••]. The full-length crystal structure of hGBP1 was determined in a nucleotide-free state [60••] and in complex with GppNHp [61]. The models exhibit many similarities to small Ras-related proteins but also several specific differences. A major structural difference is that the phosphate cap of the hGBP1–GppNHp complex (corresponding to the switch I region) shields the phosphates in the binding site from a potential GTPase-activating protein. In addition, the guanine base-site that is open to the solvent in Ras is mostly covered by an insert forming a hydrophobic pocket for the guanine base in hGBP1. It was also shown that the carboxyl terminus interacts with the amino terminus, as is the case for other members of the dynamin family. Mx proteins
Mx proteins are interferon-induced proteins found in vertebrates that exhibit strong anti-viral activity. MxA and MxB are expressed in humans, whereas Mx1, Mx2 and Mx3 are found in rodents. MxA accumulates in the cytoplasm in response to type I (α/β) interferon and inhibits multiplication of several types of RNA viruses. It forms homo-oligomers through a central interactive region and a conserved leucine zipper (LZ) element. The latter motif is responsible for intra- and intermolecular interactions and regulates both GTPase activity and viral target recognition [62,63]. Recently, it was shown that MxA oligomers interact with viral ribonucleoprotein complexes (vRNPs). This interaction retains the virus in the cytoplasm and prevents the translocation of its genome into the nucleus [64,65]. This process depends on the presence of GTPγS as a stabilizing factor [63,66], indicating that GTP binding, but not hydrolysis, is required for the antiviral activity. Apparently, GTP binding induces a conformational change that allows tight binding of the viral structures [67•]. Anti-viral activity was also detected in the presence of MxA mutants that failed to assemble and could not hydrolyze GTP in vivo, suggesting for the first time that monomers may be the active form of a dynamin-like protein [67•]. Cytoplasmic and nuclear isoforms of MxB are typically found in a granular pattern [68]. Nuclear MxB associates with heterochromatin beneath the nuclear envelope and is targeted to the nucleus through a hydrophobic ‘functional nuclear
458
Membranes and sorting
localization’ element located at the first amino acids of the amino terminus. It forms hetero-oligomers with cytoplasmic MxB through the LZ element, enabling it to enter the nucleus [69]. MxA and MxB do not form hetero-oligomers, probably due to differences in the leucine zipper sequence of the two proteins. Surprisingly, no anti-viral activity has been found for human MxB protein, and its function is still unclear. However, all the Mx proteins probably have a normal cellular function that has yet to be determined.
Conclusions Members of the dynamin family consist of a diverse group of proteins with numerous cellular locations and functions. As more members are identified, several distinct subgroups may emerge that are involved in general phenomena such as membrane fission events or anti-viral activity. A recently discovered dynamin-like protein, mG120, may represent a new class of dynamin-like proteins directly associated with the cytoskeleton. mG120, the most divergent member, contains a carboxy-terminal region that is homologous to the myosin heavy chain [70]. A common property of all the proteins of the dynamin family may be to self-assemble into higher ordered oligomers on specific templates (i.e. membranes and vRNPs). Another common property of the dynamin-like proteins may be their ability to undergo a conformational change upon GTP binding or hydrolysis, which then modifies their underlying substrate. The precise mechanism of action for these proteins is currently being pursued and the study of this family of large GTPases over the next few years promises to be very exciting and informative.
Update The first three-dimensional structure of dynamin has recently been solved using high-resolution cryo-electron microscopy (P Zhang, JE Hinshaw, unpublished data) (Figure 2b,c). The three-dimensional map of a dynamin mutant missing the PRD (∆PRD) was determined in the constricted state (+GMPPCP) and revealed a ‘T’ shaped dimer consisting of three prominent densities: leg, stalk and head (Figure 2b,c). The structure suggests that the dense stalk and head groups undergo a global conformational change upon GTP binding or hydrolysis that generates a force on the underlying lipid bilayer leading to membrane constriction. These results clearly suggest that regardless of whether dynamin acts as a molecular switch, targeting other fission components to the neck region, dynamin is clearly a force generating ‘constrictase’.
Acknowledgements We wish to thank JM Shaw for her excellent comments on the manuscript. We thank B Danino and N Dwyer for excellent technical assistance.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Hinshaw JE: Dynamin and its role in membrane fission. Annu Rev Cell Dev Biol 2000, 16:483-519.
2.
Kosaka T, Ikeda K: Possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in Drosophila. J Neurobiol 1983, 14:207-225.
3.
Schmid SL, McNiven MA, De Camilli P: Dynamin and its partners: a progress report. Curr Opin Cell Biol 1998, 10:504-512.
4.
Robinson PJ, Sontag JM, Liu JP, Fykse EM, Slaughter C, McMahon H, Sudhof TC: Dynamin GTPase regulated by protein kinase C phosphorylation in nerve terminals. Nature 1993, 365:163-166.
5.
Liu JP, Sim ATR, Robinson PJ: Calcineurin inhibition of dynamin I GTPase activity coupled to nerve terminal depolarization. Science 1994, 265:970-972.
6.
Liu JP, Powell KA, Sudhof TC, Robinson PJ: Dynamin I is a Ca2++sensitive phospholipid-binding protein with very high affinity for protein kinase C•. J Biol Chem 1994, 269:21043-21050.
7.
Takei K, Slepnev VI, Haucke V, De Camilli P: Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nat Cell Biol 1999, 1:33-39.
8.
Ringstad N, Gad H, Low P, Di Paolo G, Brodin L, Shupliakov O, De Camilli P: Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis. Neuron 1999, 24:143-154.
9.
Seedorf K, Kostka G, Lammers R, Bashkin P, Daly R, Burgess WH, van der Bliek AM, Schlessinger J, Ullrich A: Dynamin binds to SH3 domains of phospholipase C gamma and GRB-2. J Biol Chem 1994, 269:16009-16014.
10. Salim K, Bottomley MJ, Querfurth E, Zvelebil MJ, Gout I, Scaife R, Margolis RL, Gigg R, Smith CI, Driscoll PC et al.: Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton’s tyrosine kinase. EMBO J 1996, 15:6241-6250. 11. Gout I, Dhand R, Hiles ID, Fry MJ, Panayotou G, Das P, Truong O, Totty NF, Hsuan J, Booker GW: The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell 1993, 75:25-36. 12. McNiven MA, Kim L, Krueger EW, Orth JD, Cao H, Wong TW: • Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape. J Cell Biol 2000, 151:187-198. In this manuscript, dynamin 2 was localized to membrane ruffles and lamellipodia and the evidence strongly suggests dynamin interaction with cortactin in these regions. This demonstrates a direct link between dynmain and the actin cytoskeleton. 13. Hinshaw JE: Dynamin spirals. Curr Opin Struct Biol 1999, 9:260-267. 14. Sweitzer SM, Hinshaw JE: Dynamin undergoes a GTP-dependent conformational change causing vesiculation. Cell 1998, 93:1021-1029. 15. Takei K, McPherson PS, Schmid SL, De Camilli P: Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals. Nature 1995, 374:186-190. 16. Takei K, Haucke V, Slepnev V, Farsad K, Salzar M, Chen H, De Camilli P: Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes. Cell 1998, 94:131-141. 17.
Muhlberg AB, Warnock DE, Schmid SL: Domain structure and intramolecular regulation of dynamin GTPase. EMBO J 1997, 16:6676-6683.
18. Sever S, Muhlberg AB, Schmid SL: Impairment of dynamin’s GAP domain stimulates receptor-mediated endocytosis. Nature 1999, 398:481-486. 19. Smirnova E, Shurland DL, Newman-Smith ED, Pishvaee B, van der Bliek AM: A model for dynamin self-assembly based on binding between three different protein domains. J Biol Chem 1999, 274:14942-14947. 20. Sever S, Damke H, Schmid SL: Dynamin: GTP controls the •• formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis. J Cell Biol 2000, 150:1137-1148. The authors have further characterized 2 dynamin mutants in the GED region in vivo. One mutant is defective in stimulated GTPase activity and was shown to accumulate constricted coated pits. The other mutant, defective in self-assembly, increased rates of vesicle formation. From these studies and previous work, the authors suggest that dynamin acts as a classical GTPase.
Dynamin family of mechanoenzymes Danino and Hinshaw
21. Marks B, Stowell MHB, Vallis Y, Mills IG, Gibson A, Hopkins CR, • McMahon HT: GTPase activity of dynamin and resulting conformation change are essential for endocytosis. Nature 2001, 410:231-235. Using the same mutants described in [18,20••], the authors suggest in this manuscript that the GED mutants have normal stimulated GTPase activity and do not affect endocytosis. These conflicting results may be due to the different systems used to examine the mutants. 22. Bensen ES, Costaguta G, Payne GS: Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae. Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network. Genetics 2000, 154:83-97.
459
40. Wong ED, Wagner JA, Gorsich SW, McCaffery JM, Shaw JM, •• Nunnari J: The dynamin-related GTPase, Mgm1p, is an intermembrane space protein required for maintenance of fusion competent mitochondria. J Cell Biol 2000, 151:341-352. In this manuscript, the authors were surprised to observe an increase in mitochondrial fission in MGM1-deleted cells. They suggest that Mgm1p is not directly involved in fusion, instead the deletion of Mgm1p causes an uncoupling between the outer and inner mitochondrial machinery, increasing the outer mitochondrial fission events. 41. Pelloquin L, Belenguer P, Menon Y, Ducommun B: Identification of a fission yeast dynamin-related protein involved in mitochondrial DNA maintenance. Biochem Biophys Res Commun 1998, 251:720-726.
23. Luo WJ, Chang A: An endosome-to-plasma membrane pathway involved in trafficking of a mutant plasma membrane ATPase in yeast. Mol Biol Cell 2000, 11:579-592.
42. Pelloquin L, Belenguer P, Menon Y, Gas N, Ducommun B: Fission yeast Msp1 is a mitochondrial dynamin-related protein. J Cell Sci 1999, 112:4151-4161.
24. Fekkes P, Shepard KA, Yaffe MP: Gag3p, an outer membrane protein required for fission of mitochondrial tubules. J Cell Biol 2000, 151:333-340.
43. Pelloquin L, Ducommun B, Belenguer P: Interaction between the fission yeast nim1/cdr1 protein kinase and a dynamin-related protein. FEBS Lett 1999, 443:71-74.
25. Bleazard W, McCaffery JM, King EJ, Bale S, Mozdy A, Tieu Q, Nunnari J, Shaw JM: The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat Cell Biol 1999, 1:298-304.
44. Alexander C, Votruba M, Pesch UE, Thiselton DL, Mayer S, Moore A, Rodriguez M, Kellner U, Leo-Kottler B, Auburger G et al.: OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nat Genet 2000, 26:211-215.
26. Sesaki H, Jensen RE: Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape. J Cell Biol 1999, 147:699-706. 27.
Otsuga D, Keegan BR, Brisch E, Thatcher JW, Hermann GJ, Bleazard W, Shaw JM: The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast. J Cell Biol 1998, 143:333-349.
28. Smirnova E, Shurland DL, Ryazantsev SN, van der Bliek AM: A human dynamin-related protein controls the distribution of mitochondria. J Cell Biol 1998, 143:351-358. 29. Labrousse AM, Shurland DL, van der Bliek AM: Contribution of the GTPase domain to the subcellular localization of dynamin in the nematode Caenorhabditis elegans. Mol Biol Cell 1998, 9:3227-3239. 30. van der Bliek AM: A mitochondrial division apparatus takes shape. J Cell Biol 2000, 151:F1-F4. 31. Shin HW, Shinotsuka C, Torii S, Murakami K, Nakayama K: Identification and subcellular localization of a novel mammalian dynamin-related protein homologous to yeast Vps1p and Dnm1p. J Biochem (Tokyo) 1997, 122:525-30. 32. Shin HW, Takatsu H, Mukai H, Munekata E, Murakami K, Nakayama K: Intermolecular and interdomain interactions of a dynamin-related GTP-binding protein, Dnm1p/Vps1p-like protein. J Biol Chem 1999, 274:2780-2785. 33. Yoon YS, Pitts KR, Oswald BJ, McNiven MA: The dynamin-like protein (DLP1) induces membrane tubulation in living cells. Mol Biol Cell 1999, 10:314A-314A. 34. Imoto M, Tachibana I, Urrutia R: Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps1p. J Cell Sci 1998, 111:1341-1349. 35. Kamimoto T, Nagai Y, Onogi H, Muro Y, Wakabayashi T, Hagiwara M: Dymple, a novel dynamin-like high molecular weight GTPase lacking a proline-rich carboxyl-terminal domain in mammalian cells. J Biol Chem 1998, 273:1044-1051. 36. Muro Y, Kamimoto T, Tomita Y, Hagiwara M: Spectrum of autoantibodies against a dynamin-related protein, dymple. Arthritis Rheum 2000, 43:1516-1519. 37.
Hong YR, Chen CH, Cheng DS, Howng SL, Chow CC: Human dynamin-like protein interacts with the glycogen synthase kinase β. Biochem Biophys Res Commun 1998, 249:697-703. 3β
38. Jones BA, Fangman WL: Mitochondrial DNA maintenance in yeast requires a protein containing a region related to the GTP-binding domain of dynamin. Genes Dev 1992, 6:380-389. 39. Shepard KA, Yaffe MP: The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance. J Cell Biol 1999, 144:711-720.
45. Delettre C, Lenaers G, Griffoin JM, Gigarel N, Lorenzo C, Belenguer P, Pelloquin L, Grosgeorge J, Turc-Carel C, Perret E et al.: Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nat Genet 2000, 26:207-210. 46. Gu X, Verma DPS: Phragmoplastin, a dynamin-like protein associated with cell plate formation in plants. EMBO J 1996, 15:695-704. 47.
Gu X, Verma DPS: Dynamics of phragmoplastin in living cells during cell plate formation and uncoupling of cell elongation from the plane of cell division. Plant Cell 1997, 9:157-169.
48. Waizenegger I, Lukowitz W, Assaad F, Schwarz H, Jurgens G, Mayer U: The Arabidopsis KNOLLE and KEULE genes interact to promote vesicle fusion during cytokinesis. Curr Biol 2000, 10:1371-1374. 49. Zhang Z, Hong Z, Verma DPS: Phragmoplastin polymerizes into • spiral coiled structures via intermolecular interaction of two selfassembly domains. J Biol Chem 2000, 275:8779-8784. Two self-assembly domains were identified in phragmoplastin, and phragmoplastin was shown to self-assemble into higher ordered structures in low salt or upon addition of GTP-gamma-S. This further demonstrates that selfassembly is a common property among the dynamin-like proteins. 50. Hinshaw JE, Schmid SL: Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature 1995, 374:190-192. 51. Park JM, Kang SG, Pih KT, Jang HJ, Piao HL, Yoon HW, Cho MJ, Hwang I: A dynamin-like protein, ADL1, is present in membranes as a high-molecular-mass complex in Arabidopsis thaliana. Plant Physiol 1997, 115:763-771. 52. Park JM, Cho JH, Kang SG, Jang HJ, Pih KT, Piao HL, Cho MJ, Hwang I: A dynamin-like protein in Arabidopsis thaliana is involved in biogenesis of thylakoid membranes. EMBO J 1998, 17:859-867. 53. Kang SG, Jin JB, Piao HL, Jang HJ, Lim JH, Hwang I: Molecular cloning of an Arabidopsis cDNA encoding a dynamin-like protein that is localized to plastids. Plant Mol Biol 1998, 38:437-447. 54. Mikami K, Iuchi S, Yamaguchi-Shinozaki K, Shinozaki K: A novel Arabidopsis thaliana dynamin-like protein containing the pleckstrin homology domain. J Exp Bot 2000, 51:317-318. 55. Anderson SL, Carton JM, Lou J, Xing L, Rubin BY: Interferoninduced guanylate binding protein-1 (GBP-1) mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus. Virology 1999, 256:8-14. 56. Praefcke GJ, Geyer M, Schwemmle M, Robert Kalbitzer H, Herrmann C: Nucleotide-binding characteristics of human guanylate-binding protein 1 (hGBP1) and identification of the third GTP-binding motif. J Mol Biol 1999, 292:321-332.
460
57.
Membranes and sorting
Schwemmle M, Richter MF, Hermann C, Nassar N, Staeheli P: Unexpected structural requirements for GTPase activity of the interferon-induced MxA protein. J Biol Chem 1995, 270:13518-13523.
65. Weber F, Haller O, Kochs G: MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes. J Virol 2000, 74:560-563.
58. Schwemmle M, Staeheli P: The interferon-induced 67-kDa guanylate-binding protein (hGBP1) is a GTPase that converts GTP to GMP. J Biol Chem 1994, 269:11299-11305.
66. Kochs G, Haller O: GTP-bound human MxA protein interacts with the nucleocapsids of Thogoto virus (Orthomyxoviridae). J Biol Chem 1999, 274:4370-4376.
59. Neun R, Richter MF, Staeheli P, Schwemmle M: GTPase properties of the interferon-induced human guanylate-binding protein 2. FEBS Lett 1996, 390:69-72.
67. Janzen C, Kochs G, Haller O: A monomeric GTPase-negative MxA • mutant with antiviral activity. J Virol 2000, 74:8202-8206. An MxA mutant, lacking GTPase activity, was shown to retain anti-viral activity. This mutant is predicted to be defective in oligomerization and therefore suggests MxA is active in the monomeric state. This is the first dynamin-like protein predicted to be active in the monomeric form.
60. Prakash B, Praefcke GJ, Renault L, Wittinghofer A, Herrmann C: •• Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins. Nature 2000, 403:567-571. This manuscript describes the first X-ray crystal structure of a dynamin-like protein. The structure reveals a large globular GTP-binding domain with an elongated alpha-helical domain that loops back to the GTP-binding domain. 61. Prakash B, Renault L, Praefcke GJ, Herrmann C, Wittinghofer A: Triphosphate structure of guanylate-binding protein 1 and implications for nucleotide binding and GTPase mechanism. EMBO J 2000, 19:4555-4564. 62. Flohr F, SSS, Haller O, Kochs G: The central interactive region of human MxA GTPase is involved in GTPase activation and interaction with viral target structures. FEBS Lett 1999, 463:24-28. 63. Kochs G, Trost M, Janzen C, Haller O: MxA GTPase: oligomerization and GTP-dependent interaction with viral RNP target structures. Methods 1998, 15:255-263. 64. Kochs G, Haller O: Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids. Proc Natl Acad Sci USA 1999, 96:2082-2086.
68. Melen K, Keskinen P, Ronni T, Sareneva T, Lounatmaa K, Julkunen I: Human MxB protein, an interferon-alpha-inducible GTPase, contains a nuclear targeting signal and is localized in the heterochromatin region beneath the nuclear envelope. J Biol Chem 1996, 271:23478-23486. 69. Melen K, Julkunen I: Nuclear cotransport mechanism of cytoplasmic human MxB protein. J Biol Chem 1997, 272:32353-32359. 70. Kubokawa K, Miyashita T, Kubo Y: Isolation of a cDNA for a novelkDa GTP-binding protein expressed in motor neurons in the salmon brain. FEBS Lett 1998, 431:231-235.
Note added in proof The work referred to in the text as (P Zhang, JE Hinshaw, unpublished data) is now in press: 71. Zhang P, Hinshaw JE: Three dimensional reconstruction of dynamin in the constricted state. Nat Cell Biol 2001, in press.