Three-dimensional Map of a Plant V-ATPase Based on Electron Microscopy
2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês
10.1074/jbc.m112011200
ISSN1083-351X
AutoresInes Domgall, David Venzke, Ulrich Lüttge, Rafael Ratajczak, Bettina Böttcher,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoV-ATPases pump protons into the interior of various subcellular compartments at the expense of ATP. Previous studies have shown that these pumps comprise a membrane-integrated, proton-translocating (V0), and a soluble catalytic (V1) subcomplex connected to one another by a thin stalk region. We present two three-dimensional maps derived from electron microscopic images of the complete V-ATPase complex from the plant Kalanchoë daigremontiana at a resolution of 2.2 nm. In the presence of a non-hydrolyzable ATP analogue, the details of the stalk region between V0 and V1 were revealed for the first time in their three-dimensional organization. A central stalk was surrounded by three peripheral stalks of different sizes and shapes. In the absence of the ATP analogue, the tilt of V0changed with respect to V1, and the stalk region was less clearly defined, perhaps due to increased flexibility and partial detachment of some of the peripheral stalks. These structural changes corresponded to decreased stability of the complex and might be the initial step in a controlled disassembly. V-ATPases pump protons into the interior of various subcellular compartments at the expense of ATP. Previous studies have shown that these pumps comprise a membrane-integrated, proton-translocating (V0), and a soluble catalytic (V1) subcomplex connected to one another by a thin stalk region. We present two three-dimensional maps derived from electron microscopic images of the complete V-ATPase complex from the plant Kalanchoë daigremontiana at a resolution of 2.2 nm. In the presence of a non-hydrolyzable ATP analogue, the details of the stalk region between V0 and V1 were revealed for the first time in their three-dimensional organization. A central stalk was surrounded by three peripheral stalks of different sizes and shapes. In the absence of the ATP analogue, the tilt of V0changed with respect to V1, and the stalk region was less clearly defined, perhaps due to increased flexibility and partial detachment of some of the peripheral stalks. These structural changes corresponded to decreased stability of the complex and might be the initial step in a controlled disassembly. vacuolar H+-transporting adenosine triphosphatase F0F1-ATPase adenosine[5′-β,γ-imido]triphosphate V-ATPases1 are found in all eukaryotic cells. They hydrolyze ATP to pump protons into various intracellular compartments (1.Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (520) Google Scholar). In plant tonoplasts the proton motive force generated by the V-ATPase is used for secondary transport processes contributing to osmoregulation, ion and pH homeostasis, nutrient and remnant storage, and plant defense (2.Taiz L. J. Exp. Biol. 1992; 172: 113-122Crossref PubMed Google Scholar).V-ATPases are highly conserved among species, and their gross architecture is similar to that of the well characterized F-ATPases. In the V-ATPases, the soluble V1 subcomplex is known to carry the catalytic nucleotide-binding sites and to be connected via a thinner stalk region to the membrane-integrated V0subcomplex, which contains the proton-translocating machinery. The exact subunit composition and stoichiometry of V-ATPases, however, is still controversial. In yeast, the V1 subcomplex is probably formed by the subunits (AB)3, C—H, and the V0 subcomplex by the subunits c, c′, c", a, and d (for review see Ref. 3.Forgac M. J. Biol. Chem. 1999; 274: 12951-12954Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Homologues of the c′- and c"-subunits have not been identified in plants as of yet (4.Sze H. Li X. Palmgren M.G. Plant Cell. 1999; 11: 677-689PubMed Google Scholar).Among the subunits known to comprise V-ATPases, several share significant sequence homology to subunits of F-ATPases. The catalytic A-subunits of V-ATPase are homologous to the catalytic β-subunits in F-ATPase (5.Zimniak L. Dittrich P. Gogarten J.P. Kibak H. Taiz L. J. Biol. Chem. 1988; 263: 9102-9112Abstract Full Text PDF PubMed Google Scholar) and the B-subunits of V-ATPase to the non-catalytic α-subunits in F-ATPase (6.Nelson H. Mandiyan S. Nelson N. J. Biol. Chem. 1989; 264: 1775-1778Abstract Full Text PDF PubMed Google Scholar). The membrane-integrated V-ATPase c-subunit has probably emerged by gene duplication (7.Mandel M. Moriyama Y. Hulmes J.D. Pan Y.C. Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5521-5524Crossref PubMed Scopus (238) Google Scholar) from a common ancestor of F- and V-ATPases. The G-subunit of V-ATPases has sequence similarity to the hydrophilic part of the membrane-anchored F-ATPase b-subunit (8.Supekova L. Sbia M. Supek F. Ma Y. Nelson N. J. Exp. Biol. 1996; 199: 1147-1156Crossref PubMed Google Scholar). Other components of the F-ATPase machinery do not have any homologues in V-ATPase. Furthermore, V-ATPases contain various subunits (C, F, H, a, and d) whose functions and relationships to F-ATPases still need to be elucidated. This divergence might reflect an adaptation to the different physiological requirements of F- and V-ATPases. Unlike F-ATPases, V-ATPases usually do not synthesize ATP but hydrolyze ATP to generate proton motive force. When resources become scarce, V-ATPases are required to be shut down to save the diminishing ATP levels for more vital cellular processes. This shutdown is achieved by reversible disassembly of the V-ATPase complex, a process that is unknown in the regulation of F-ATPases. In yeast, disassembly is initiated in response to glucose deprivation (for review see Ref. 9.Kane P.M. Parra K.J. J. Exp. Biol. 2000; 203: 81-87Crossref PubMed Google Scholar). Although the underlying molecular mechanism is not completely understood, it is speculated that the disassembly might be initiated by a brief drop in cellular ATP levels caused by decreasing glucose levels (10.Parra K.J. Kane P.M. Mol. Cell. Biol. 1998; 18: 7064-7074Crossref PubMed Google Scholar). This drop in ATP levels possibly causes conformational changes in the V-ATPase that start the disassembly process.Although we have detailed knowledge of the structural organization of various subcomplexes of F-ATPases (11.Stock D. Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1078) Google Scholar, 12.Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar), only little is known about the structure of V-ATPases. The V-ATPase topology was investigated by a number of biochemical experiments, particularly contacts and proximities between subunits were explored by cross-linking studies (for review see Refs. 13.Graham L.A. Powell B. Stevens T.H. J. Exp. Biol. 2000; 203: 61-70Crossref PubMed Google Scholar and 14.Grüber G. Wieczorek H. Harvey W.R. Müller V. J. Exp. Biol. 2001; 204: 2597-2605Crossref PubMed Google Scholar). These topological data were complemented by low resolution three-dimensional information about the isolated V1 subcomplex (15.Svergun D.I. Konrad S. Huss M. Koch M.H. Wieczorek H. Altendorf K. Volkov V.V. Grüber G. Biochemistry. 1998; 37: 17659-17663Crossref PubMed Scopus (56) Google Scholar, 16.Grüber G. Radermacher M. Ruiz T. Godovac-Zimmermann J. Canas B. Kleine-Kohlbrecher D. Huss M. Harvey W.R. Wieczorek H. Biochemistry. 2000; 39: 8609-8616Crossref PubMed Scopus (69) Google Scholar, 17.Radermacher M. Ruiz T. Wieczorek H. Grüber G. J. Struct. Biol. 2001; 135: 26-37Crossref PubMed Scopus (94) Google Scholar) and the isolated V0 subcomplex (18.Wilkens S. Forgac M. J. Biol. Chem. 2001; 276: 44064-44068Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar).Up to now, three-dimensional information for a complete V-ATPase complex has not been available. Two-dimensional projection maps derived from electron micrographs of two different V-ATPases (19.Ubbink-Kok T. Boekema E.J. van Breemen J.F. Brisson A. Konings W.N. Lolkema J.S. J. Mol. Biol. 2000; 296: 311-321Crossref PubMed Scopus (27) Google Scholar, 20.Wilkens S. Vasilyeva E. Forgac M. J. Biol. Chem. 1999; 274: 31804-31810Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) have given an impression of the overall architecture of the enzyme and revealed a complicated connecting region, composed of at least three stalks, between V0 and V1 (21.Boekema E.J. Ubbink-Kok T. Lolkema J.S. Brisson A. Konings W.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14291-14293Crossref PubMed Scopus (91) Google Scholar, 22.Boekema E.J. van Breemen J.F. Brisson A. Ubbink-Kok T. Konings W.N. Lolkema J.S. Nature. 1999; 401: 37-38Crossref PubMed Scopus (76) Google Scholar). However, the lack of three-dimensional information has made it impossible to come to any conclusions on the exact number of connecting elements or to determine whether these peripheral connections are formed by structurally identical components.To shed some light on the architecture of V-ATPases, we have calculated three-dimensional maps from electron micrographs of the gold thioglucose-stained plant V-ATPase from Kalanchoëdaigremontiana (Mother-of-Thousands) in the presence and absence of the non-hydrolyzable ATP analogue AMP-PNP. AMP-PNP served to mimic nucleotide concentrations comparable with those found in cells under normal growth conditions, whereas the V-ATPase without AMP-PNP added reflects a complex under complete nucleotide-deprived surroundings. With these studies we were able to get detailed information of the three-dimensional organization of the V-ATPase complex as well as of its response to changing nucleotide concentrations.RESULTSThe K. daigremontiana V-ATPase samples used in our analysis by electron microscopy were characterized for purity and activity by SDS-PAGE and activity assays, respectively. Fig. 1A shows the typical polypeptide pattern of the V-ATPase after purification that corresponded to what has been reported previously (23.Ratajczak R. Kemna I. Lüttge U. Planta. 1994; 195: 226-236Crossref Scopus (24) Google Scholar, 33.Warren M. Smith J.A.C. Apps D.K. Biochim. Biophys. Acta. 1992; 1106: 117-125Crossref PubMed Scopus (24) Google Scholar). During recent work the D-subunit (34 kDa) and two E-subunit isoforms (32 and 33 kDa) have been identified in the K. daigremontianaV-ATPase by matrix-assisted laser desorption ionization-mass spectroscopy (34.Ratajczak, R., Pfeifer, T., Drobny, M., Schnölzer, M., and Lüttge, U. (2002) Biol. Plantarum (Prague), in pressGoogle Scholar). All other subunits of V1 (A–C and F–H) and subunits a, c, and d of V0 were identified according to their molecular mass in comparison to the molecular mass of V-ATPase subunits of other species (for review see Ref. 3.Forgac M. J. Biol. Chem. 1999; 274: 12951-12954Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The activity of the preparation was 1.7 μmol of ATP mg−1 protein min−1 and was completely inhibited by the specific V-ATPase inhibitor concanamycin A1. Samples for electron microscopy were prepared from as little as 40 ng. Electron micrographs of V-ATPase negatively stained with gold thioglucose showed a homogeneous particle distribution (Fig. 1B). For image processing, only particle images of dumbbell-like shape were chosen to ensure that all of the selected complexes consisted of V0and V1.V-ATPase with AMP-PNP AddedFor imaging the V-ATPase in the presence of AMP-PNP, AMP-PNP (final concentration 2 mm) was added to both the purified enzyme and to the staining solution. We provided the substrate analogue AMP-PNP both to mimic nucleotide concentrations comparable with those found in cells under normal growth conditions and to ensure a stable, active conformation of the enzyme complex from a structural point of view. From a total of 134 micrographs (of which 59 were tilted by either 20, 25, or 30°) of V-ATPase, almost 12,000 single particle images were extracted and subjected to objective alignment procedures followed by multivariate statistical analysis. The class averages that were obtained represented different projections of the V-ATPase (Fig. 2A). The lower V0subcomplex was bean-shaped, and the upper V1 subcomplex appeared to be asymmetric, with a prominent "spike" density on top (Fig. 2A, white arrowheads). In addition, small "knob"-like densities were visible at the periphery of V1 (Fig. 2A, 1–3, white arrows). V1 and V0 were connected by a central stalk from which a strong peripheral density extended in most of the class averages (Fig. 2A, asterisks). Some projections revealed an additional elongated second peripheral density (Fig. 2A, 1, 2, and 4, black arrows) that stretched out from V0 and reached all the way up to the top of V1.Figure 2Two-dimensional projection maps and different representations of the three-dimensional map of V-ATPase with addedAMP-PNP. A, selected class averages. B, surface representation of the final three-dimensional map viewed in the same directions as calculated for the projection directions of the class averages. C, projections of the three-dimensional map into the directions of the class averages shown in A. The respective projection angles are given in the bottom panel. Labels: white arrowheads, spike; white arrows, knobs; asterisks, prominent peripheral stalk density;black arrows, faint connection. Bar = 10 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The class averages were combined into a three-dimensional map shown as surface representations in Fig. 2B. Projections of the three-dimensional map (Fig. 2C) were calculated in the same directions as determined for the class averages to validate the map. The projections matched the corresponding class averages (Fig. 2A), illustrating that the three-dimensional map described the original data accurately. This was further supported by the Fourier shell correlation computed from two separate three-dimensional maps each calculated from half of the collected data (Fig. 3, curve 1). The Fourier shell correlation showed a smooth fall off with increasing Fourier spacing, dropping to a correlation of 0.5 at 1/2.2 nm−1 and traversing the 3ς curve at 1/1.6 nm−1 (limit introduced by bandpass filtering of the original data).The density distribution inside the complex can be better explored in slices through the map (Fig. 4A) in an orientation approximately perpendicular to the supposed plane of the membrane. These slices revealed a V1 with distinct structures, a V0 with an oval cross-section, and a thin stalk region. The V1 had an overall diameter of 11.7 nm and an elongated narrow cavity in its center. At the top of V1, the spike density (Fig. 4A, slices 3–10, white arrowheads) was visible in half of the slices, indicating an elongated structure. The V0 measured 12.1 nm in the supposed plane of the membrane and 8.1 nm perpendicular to it. It had a dense outer shell that surrounded a structured cavity inside (Fig. 4A, slices 5–9). The gap of 4.4 nm between V1 and V0 was bridged by four distinct stalks, one central and three peripheral. The central stalk had a maximal diameter of 3.6 nm (Fig. 4A, slices 5–9). The peripheral stalks varied in their diameters and linked peripheral parts of V1 to more central components in V0, near the point of intersection of the central stalk with V0. The most prominent of these peripheral stalks had a diameter of 4.9 nm (Fig. 4A, slices 11–15,black open arrowheads), the intermediate one 3.6 nm (Fig. 4A, slices 5–8, white open arrowheads), and the faintest stalk 2.4 nm (Fig. 4A, slices 6–8, black arrowheads). In the following, the described peripheral stalks are referred to as "prominent," "intermediate," and "faint" stalk.Figure 4Slices through the three-dimensional map of the V-ATPase with added AMP-PNP. A, slices (0.8 nm thick) perpendicular to the supposed plane of the membrane. B, selected slices (0.4 nm thick) parallel to the supposed plane of the membrane at the positions indicated in A (slice 10). Labels: black arrows, A-subunits;white arrows, B-subunits; white arrowheads, spike; black arrowheads, faint stalk; open black arrowheads, prominent stalk; open white arrowheads, intermediate stalk. Bar = 10 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In Fig. 4B, a selection of slices through the three-dimensional map parallel to the supposed plane of the membrane is shown. The first section revealed the spike at the crest of V1 to have the form of a circular arc fragment of about 100°. Section two showed a cross-section through the upper part of V1. The likely positions of the three A- and three B-subunits are indicated by black and white arrows, respectively (see "Discussion"). Section three described the transition from V1 to the stalk region, with a rather round outline. Section four showed cross-sections of the central and the three peripheral stalks (see arrowheads). The positions of the peripheral stalks could be described by the angles enclosed between the axes connecting the centers of the peripheral stalks to the midpoint of the central stalk. These angles were 60° between the prominent and the intermediate stalks, 120° between the prominent and the faint stalks, and 180° between the faint and intermediate stalks. Section five represented a cross-section of the V0. It showed a pronounced outer ring of density and a diffuse density distribution inside.V-ATPase without AMP-PNP AddedDuring purification loosely bound nucleotides were removed from the V-ATPase. To test the stability of the complex within different nucleotide contexts, in a separate experiment the purified V-ATPase was reapplied to a DEAE-cellulose column in the presence or absence of the substrate analogue AMP-PNP. When AMP-PNP was not added to the complex, subunits came off in wash fractions before the elution (250 mm NaCl) as seen on SDS-PAGE, and almost no intact particles were observed by electron microscopy (data not shown). In contrast, when AMP-PNP was added to the sample and buffers, the majority of the protein that was eluted from the column represented intact V-ATPase holoenzymes as seen on SDS-PAGE and as visualized by electron microscopy (data not shown).To investigate what structural changes were linked to the observed lability of the complex in the absence of loosely bound nucleotides,i.e. without added nucleotides in the surrounding buffer, we analyzed another data set of about 10,000 individual images of the same purified V-ATPase preparation as used in the electron microscopic investigation described above, but this time without adding AMP-PNP. The data were collected and processed in the same way as the first data set. A surface representation of the three-dimensional map (Fig. 5B) shows the same gross architecture as already observed in the three-dimensional map of the V-ATPase with AMP-PNP added (Fig. 5A, hereafter referred to as AMP-PNP map), although some of the structural details were different. Both V1 and V0 occupied the same volumes as in the AMP-PNP map. Again, V1 had a spike at its very top (see white arrowhead), albeit less prominent than in the AMP-PNP map. V1 and V0 were also joined by a central stalk that was thinner than that in the AMP-PNP map. Furthermore, a peripheral density extended from the base of the central stalk like a little arm. When the three-dimensional map was aligned to the AMP-PNP map with the spike and the direction of elongation of the V0 subcomplex in the plane of the membrane matching, this small stalk lined up with the faint stalk in the AMP-PNP map. In the class averages and in slices through the three-dimensional map (not shown), the typical features of the three peripheral stalks were visible but were indistinct and lower in density than in the AMP-PNP map and were therefore not seen in the surface representation of the three-dimensional map. The most obvious difference between the two maps, however, was the tilt of V0 with respect to V1 which changed by about 30° in comparison to the AMP-PNP map.Figure 5Surface representation of the three-dimensional maps of the V-ATPase. A, with AMP-PNP added; B, without AMP-PNP. White arrowheads label the spike. Bar = 10 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) V-ATPases1 are found in all eukaryotic cells. They hydrolyze ATP to pump protons into various intracellular compartments (1.Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (520) Google Scholar). In plant tonoplasts the proton motive force generated by the V-ATPase is used for secondary transport processes contributing to osmoregulation, ion and pH homeostasis, nutrient and remnant storage, and plant defense (2.Taiz L. J. Exp. Biol. 1992; 172: 113-122Crossref PubMed Google Scholar). V-ATPases are highly conserved among species, and their gross architecture is similar to that of the well characterized F-ATPases. In the V-ATPases, the soluble V1 subcomplex is known to carry the catalytic nucleotide-binding sites and to be connected via a thinner stalk region to the membrane-integrated V0subcomplex, which contains the proton-translocating machinery. The exact subunit composition and stoichiometry of V-ATPases, however, is still controversial. In yeast, the V1 subcomplex is probably formed by the subunits (AB)3, C—H, and the V0 subcomplex by the subunits c, c′, c", a, and d (for review see Ref. 3.Forgac M. J. Biol. Chem. 1999; 274: 12951-12954Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Homologues of the c′- and c"-subunits have not been identified in plants as of yet (4.Sze H. Li X. Palmgren M.G. Plant Cell. 1999; 11: 677-689PubMed Google Scholar). Among the subunits known to comprise V-ATPases, several share significant sequence homology to subunits of F-ATPases. The catalytic A-subunits of V-ATPase are homologous to the catalytic β-subunits in F-ATPase (5.Zimniak L. Dittrich P. Gogarten J.P. Kibak H. Taiz L. J. Biol. Chem. 1988; 263: 9102-9112Abstract Full Text PDF PubMed Google Scholar) and the B-subunits of V-ATPase to the non-catalytic α-subunits in F-ATPase (6.Nelson H. Mandiyan S. Nelson N. J. Biol. Chem. 1989; 264: 1775-1778Abstract Full Text PDF PubMed Google Scholar). The membrane-integrated V-ATPase c-subunit has probably emerged by gene duplication (7.Mandel M. Moriyama Y. Hulmes J.D. Pan Y.C. Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5521-5524Crossref PubMed Scopus (238) Google Scholar) from a common ancestor of F- and V-ATPases. The G-subunit of V-ATPases has sequence similarity to the hydrophilic part of the membrane-anchored F-ATPase b-subunit (8.Supekova L. Sbia M. Supek F. Ma Y. Nelson N. J. Exp. Biol. 1996; 199: 1147-1156Crossref PubMed Google Scholar). Other components of the F-ATPase machinery do not have any homologues in V-ATPase. Furthermore, V-ATPases contain various subunits (C, F, H, a, and d) whose functions and relationships to F-ATPases still need to be elucidated. This divergence might reflect an adaptation to the different physiological requirements of F- and V-ATPases. Unlike F-ATPases, V-ATPases usually do not synthesize ATP but hydrolyze ATP to generate proton motive force. When resources become scarce, V-ATPases are required to be shut down to save the diminishing ATP levels for more vital cellular processes. This shutdown is achieved by reversible disassembly of the V-ATPase complex, a process that is unknown in the regulation of F-ATPases. In yeast, disassembly is initiated in response to glucose deprivation (for review see Ref. 9.Kane P.M. Parra K.J. J. Exp. Biol. 2000; 203: 81-87Crossref PubMed Google Scholar). Although the underlying molecular mechanism is not completely understood, it is speculated that the disassembly might be initiated by a brief drop in cellular ATP levels caused by decreasing glucose levels (10.Parra K.J. Kane P.M. Mol. Cell. Biol. 1998; 18: 7064-7074Crossref PubMed Google Scholar). This drop in ATP levels possibly causes conformational changes in the V-ATPase that start the disassembly process. Although we have detailed knowledge of the structural organization of various subcomplexes of F-ATPases (11.Stock D. Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1078) Google Scholar, 12.Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar), only little is known about the structure of V-ATPases. The V-ATPase topology was investigated by a number of biochemical experiments, particularly contacts and proximities between subunits were explored by cross-linking studies (for review see Refs. 13.Graham L.A. Powell B. Stevens T.H. J. Exp. Biol. 2000; 203: 61-70Crossref PubMed Google Scholar and 14.Grüber G. Wieczorek H. Harvey W.R. Müller V. J. Exp. Biol. 2001; 204: 2597-2605Crossref PubMed Google Scholar). These topological data were complemented by low resolution three-dimensional information about the isolated V1 subcomplex (15.Svergun D.I. Konrad S. Huss M. Koch M.H. Wieczorek H. Altendorf K. Volkov V.V. Grüber G. Biochemistry. 1998; 37: 17659-17663Crossref PubMed Scopus (56) Google Scholar, 16.Grüber G. Radermacher M. Ruiz T. Godovac-Zimmermann J. Canas B. Kleine-Kohlbrecher D. Huss M. Harvey W.R. Wieczorek H. Biochemistry. 2000; 39: 8609-8616Crossref PubMed Scopus (69) Google Scholar, 17.Radermacher M. Ruiz T. Wieczorek H. Grüber G. J. Struct. Biol. 2001; 135: 26-37Crossref PubMed Scopus (94) Google Scholar) and the isolated V0 subcomplex (18.Wilkens S. Forgac M. J. Biol. Chem. 2001; 276: 44064-44068Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Up to now, three-dimensional information for a complete V-ATPase complex has not been available. Two-dimensional projection maps derived from electron micrographs of two different V-ATPases (19.Ubbink-Kok T. Boekema E.J. van Breemen J.F. Brisson A. Konings W.N. Lolkema J.S. J. Mol. Biol. 2000; 296: 311-321Crossref PubMed Scopus (27) Google Scholar, 20.Wilkens S. Vasilyeva E. Forgac M. J. Biol. Chem. 1999; 274: 31804-31810Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) have given an impression of the overall architecture of the enzyme and revealed a complicated connecting region, composed of at least three stalks, between V0 and V1 (21.Boekema E.J. Ubbink-Kok T. Lolkema J.S. Brisson A. Konings W.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14291-14293Crossref PubMed Scopus (91) Google Scholar, 22.Boekema E.J. van Breemen J.F. Brisson A. Ubbink-Kok T. Konings W.N. Lolkema J.S. Nature. 1999; 401: 37-38Crossref PubMed Scopus (76) Google Scholar). However, the lack of three-dimensional information has made it impossible to come to any conclusions on the exact number of connecting elements or to determine whether these peripheral connections are formed by structurally identical components. To shed some light on the architecture of V-ATPases, we have calculated three-dimensional maps from electron micrographs of the gold thioglucose-stained plant V-ATPase from Kalanchoëdaigremontiana (Mother-of-Thousands) in the presence and absence of the non-hydrolyzable ATP analogue AMP-PNP. AMP-PNP served to mimic nucleotide concentrations comparable with those found in cells under normal growth conditions, whereas the V-ATPase without AMP-PNP added reflects a complex under complete nucleotide-deprived surroundings. With these studies we were able to get detailed information of the three-dimensional organization of the V-ATPase complex as well as of its response to changing nucleotide concentrations. RESULTSThe K. daigremontiana V-ATPase samples used in our analysis by electron microscopy were characterized for purity and activity by SDS-PAGE and activity assays, respectively. Fig. 1A shows the typical polypeptide pattern of the V-ATPase after purification that corresponded to what has been reported previously (23.Ratajczak R. Kemna I. Lüttge U. Planta. 1994; 195: 226-236Crossref Scopus (24) Google Scholar, 33.Warren M. Smith J.A.C. Apps D.K. Biochim. Biophys. Acta. 1992; 1106: 117-125Crossref PubMed Scopus (24) Google Scholar). During recent work the D-subunit (34 kDa) and two E-subunit isoforms (32 and 33 kDa) have been identified in the K. daigremontianaV-ATPase by matrix-assisted laser desorption ionization-mass spectroscopy (34.Ratajczak, R., Pfeifer, T., Drobny, M., Schnölzer, M., and Lüttge, U. (2002) Biol. Plantarum (Prague), in pressGoogle Scholar). All other subunits of V1 (A–C and F–H) and subunits a, c, and d of V0 were identified according to their molecular mass in comparison to the molecular mass of V-ATPase subunits of other species (for review see Ref. 3.Forgac M. J. Biol. Chem. 1999; 274: 12951-12954Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The activity of the preparation was 1.7 μmol of ATP mg−1 protein min−1 and was completely inhibited by the specific V-ATPase inhibitor concanamycin A1. Samples for electron microscopy were prepared from as little as 40 ng. Electron micrographs of V-ATPase negatively stained with gold thioglucose showed a homogeneous particle distribution (Fig. 1B). For image processing, only particle images of dumbbell-like shape were chosen to ensure that all of the selected complexes consisted of V0and V1.V-ATPase with AMP-PNP AddedFor imaging the V-ATPase in the presence of AMP-PNP, AMP-PNP (final concentration 2 mm) was added to both the purified enzyme and to the staining solution. We provided the substrate analogue AMP-PNP both to mimic nucleotide concent
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