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Biochemical and biophysical properties of interactions between subunits of the peripheral stalk region of human V-ATPase.

Rahman S, Yamato I, Saijo S, Mizutani K, Ishizuka-Katsura Y, Ohsawa N, Terada T, Shirouzu M, Yokoyama S, Iwata S, Murata T - PLoS ONE (2013)

Bottom Line: The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis.We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities.No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, Tokyo University of Science, Chiba, Japan. srahman@rs.tus.ac.jp

ABSTRACT
Peripheral stalk subunits of eukaryotic or mammalian vacuolar ATPases (V-ATPases) play key roles in regulating its assembly and disassembly. In a previous study, we purified several subunits and their isoforms of the peripheral stalk region of Homo sapiens (human) V-ATPase; such as C1, E1G1, H, and the N-terminal cytoplasmic region of V(o), a1. Here, we investigated the in vitro binding interactions of the subunits at the stalk region and measured their specific affinities. Surface plasmon resonance experiments revealed that the subunit C1 binds the E1G1 heterodimer with both high and low affinities (2.8 nM and 1.9 µM, respectively). In addition, an E1G1-H complex can be formed with high affinity (48 nM), whereas affinities of other subunit pairs appeared to be low (∼0.21-3.0 µM). The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis. We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities. No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly. Multiple interactions of variable affinity in the stalk region may be important to the mechanism of reversible dissociation of the intact V-ATPase.

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Ternary interactions of E1G1, C1, and H.(A) Model of E1G1-C1-H assembly. Dotted arrows (red) and solid arrows (black) indicate weak and strong binding, respectively. (B) Gel filtration profile of the H/C1/E1G1 mixture (red) in comparison to H (purple), E1G1 (green), and C1 (blue) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Border colors indicate samples corresponding to the color scheme used in 4B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of the H/C1/E1G1 mixture. A molar ratio of 3∶1∶1 of E1G1:C1:H proteins was prepared and incubated on ice for 1 h (lane 4). Bands corresponding to one molar amount of E1G1, C1, and H proteins are visible in lanes 1, 2, and 3, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the E1G1-C1-H mixture band eluted from the native gel in panel X (lane 4). (E) SDS-PAGE of the eluted proteins from the His-tag pulldown experiment. Lane 1, fraction eluted using buffer B; lane 2, subunits bound with His-tagged H subunit eluted using buffer C.
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pone-0055704-g004: Ternary interactions of E1G1, C1, and H.(A) Model of E1G1-C1-H assembly. Dotted arrows (red) and solid arrows (black) indicate weak and strong binding, respectively. (B) Gel filtration profile of the H/C1/E1G1 mixture (red) in comparison to H (purple), E1G1 (green), and C1 (blue) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Border colors indicate samples corresponding to the color scheme used in 4B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of the H/C1/E1G1 mixture. A molar ratio of 3∶1∶1 of E1G1:C1:H proteins was prepared and incubated on ice for 1 h (lane 4). Bands corresponding to one molar amount of E1G1, C1, and H proteins are visible in lanes 1, 2, and 3, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the E1G1-C1-H mixture band eluted from the native gel in panel X (lane 4). (E) SDS-PAGE of the eluted proteins from the His-tag pulldown experiment. Lane 1, fraction eluted using buffer B; lane 2, subunits bound with His-tagged H subunit eluted using buffer C.

Mentions: To determine higher-order binding among subunits, ternary and quaternary binding interactions were examined. Interestingly, complex formation was not observed using equimolar amounts of E1G1, C1, and H, with the H subunit being expelled, appearing as two peaks in the gel filtration analysis (–B) (model shown in Figure 4A). We expected that by using a 3∶1∶1 molar ratio of E1G1:C1:H, a stable CE3G3H putative ternary complex would be formed. Unexpectedly, however, in gel filtration, this mixture of proteins appeared as single peak at 1.85 ml (Figure 4B–C), a position similar to that of C1-E1G1 and H-E1G1 complexes (Figure 2B–C and Figure 3B–C). The binding of one E1G1 with the C1foot region was not sufficiently strong for it to remain in the complex to form CE3G3H; instead it remained unbound, leading to the formation of a C1(E1G1)2H complex. We interpreted that the E1G1-C1-H putative ternary complex was not formed because of the lower binding affinities between C1-H and E1G1-C1foot; thus, we thought that the peak in gel filtration contained a mixture of two strong complexes (E1G1-C1head and E1G1-H). The integrity of the putative ternary complex (E1G1-C1-H) formation was further examined by basic native PAGE, by mixing a similar molar ratio of each protein as for gel filtration (Figure 4D). In comparison to subunit C1, E1G1, or H, the migration rate of the (E1G1-C1-H) complex was delayed. The band corresponding to the complex was excised from the native gel and analyzed by SDS-PAGE (Figure 4D; Panel Y), with results consistent with the existence of a mixture of E1G1-C1 and E1G1-H complexes. A pulldown assay using His-tagged-H subunit showed strong binding with E1G1 but weak binding of C1, suggesting no stable putative ternary complex was formed (Figure 4E, lane 2). We speculate that two binary complexes (E1G1C1 and E1G1H) are sufficiently strong, together with other weak binding interactions, to form the L-shaped structure of CE3G3H in vivo[27].


Biochemical and biophysical properties of interactions between subunits of the peripheral stalk region of human V-ATPase.

Rahman S, Yamato I, Saijo S, Mizutani K, Ishizuka-Katsura Y, Ohsawa N, Terada T, Shirouzu M, Yokoyama S, Iwata S, Murata T - PLoS ONE (2013)

Ternary interactions of E1G1, C1, and H.(A) Model of E1G1-C1-H assembly. Dotted arrows (red) and solid arrows (black) indicate weak and strong binding, respectively. (B) Gel filtration profile of the H/C1/E1G1 mixture (red) in comparison to H (purple), E1G1 (green), and C1 (blue) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Border colors indicate samples corresponding to the color scheme used in 4B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of the H/C1/E1G1 mixture. A molar ratio of 3∶1∶1 of E1G1:C1:H proteins was prepared and incubated on ice for 1 h (lane 4). Bands corresponding to one molar amount of E1G1, C1, and H proteins are visible in lanes 1, 2, and 3, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the E1G1-C1-H mixture band eluted from the native gel in panel X (lane 4). (E) SDS-PAGE of the eluted proteins from the His-tag pulldown experiment. Lane 1, fraction eluted using buffer B; lane 2, subunits bound with His-tagged H subunit eluted using buffer C.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569449&req=5

pone-0055704-g004: Ternary interactions of E1G1, C1, and H.(A) Model of E1G1-C1-H assembly. Dotted arrows (red) and solid arrows (black) indicate weak and strong binding, respectively. (B) Gel filtration profile of the H/C1/E1G1 mixture (red) in comparison to H (purple), E1G1 (green), and C1 (blue) monomers. (C) SDS-PAGE analysis of the eluted fractions from gel filtration chromatography. Border colors indicate samples corresponding to the color scheme used in 4B. “C” indicates control proteins. (D) Panel X: Basic native polyacrylamide gel electrophoresis analysis of the H/C1/E1G1 mixture. A molar ratio of 3∶1∶1 of E1G1:C1:H proteins was prepared and incubated on ice for 1 h (lane 4). Bands corresponding to one molar amount of E1G1, C1, and H proteins are visible in lanes 1, 2, and 3, respectively. Panel Y: SDS-PAGE (12% gel) analysis of the E1G1-C1-H mixture band eluted from the native gel in panel X (lane 4). (E) SDS-PAGE of the eluted proteins from the His-tag pulldown experiment. Lane 1, fraction eluted using buffer B; lane 2, subunits bound with His-tagged H subunit eluted using buffer C.
Mentions: To determine higher-order binding among subunits, ternary and quaternary binding interactions were examined. Interestingly, complex formation was not observed using equimolar amounts of E1G1, C1, and H, with the H subunit being expelled, appearing as two peaks in the gel filtration analysis (–B) (model shown in Figure 4A). We expected that by using a 3∶1∶1 molar ratio of E1G1:C1:H, a stable CE3G3H putative ternary complex would be formed. Unexpectedly, however, in gel filtration, this mixture of proteins appeared as single peak at 1.85 ml (Figure 4B–C), a position similar to that of C1-E1G1 and H-E1G1 complexes (Figure 2B–C and Figure 3B–C). The binding of one E1G1 with the C1foot region was not sufficiently strong for it to remain in the complex to form CE3G3H; instead it remained unbound, leading to the formation of a C1(E1G1)2H complex. We interpreted that the E1G1-C1-H putative ternary complex was not formed because of the lower binding affinities between C1-H and E1G1-C1foot; thus, we thought that the peak in gel filtration contained a mixture of two strong complexes (E1G1-C1head and E1G1-H). The integrity of the putative ternary complex (E1G1-C1-H) formation was further examined by basic native PAGE, by mixing a similar molar ratio of each protein as for gel filtration (Figure 4D). In comparison to subunit C1, E1G1, or H, the migration rate of the (E1G1-C1-H) complex was delayed. The band corresponding to the complex was excised from the native gel and analyzed by SDS-PAGE (Figure 4D; Panel Y), with results consistent with the existence of a mixture of E1G1-C1 and E1G1-H complexes. A pulldown assay using His-tagged-H subunit showed strong binding with E1G1 but weak binding of C1, suggesting no stable putative ternary complex was formed (Figure 4E, lane 2). We speculate that two binary complexes (E1G1C1 and E1G1H) are sufficiently strong, together with other weak binding interactions, to form the L-shaped structure of CE3G3H in vivo[27].

Bottom Line: The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis.We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities.No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, Tokyo University of Science, Chiba, Japan. srahman@rs.tus.ac.jp

ABSTRACT
Peripheral stalk subunits of eukaryotic or mammalian vacuolar ATPases (V-ATPases) play key roles in regulating its assembly and disassembly. In a previous study, we purified several subunits and their isoforms of the peripheral stalk region of Homo sapiens (human) V-ATPase; such as C1, E1G1, H, and the N-terminal cytoplasmic region of V(o), a1. Here, we investigated the in vitro binding interactions of the subunits at the stalk region and measured their specific affinities. Surface plasmon resonance experiments revealed that the subunit C1 binds the E1G1 heterodimer with both high and low affinities (2.8 nM and 1.9 µM, respectively). In addition, an E1G1-H complex can be formed with high affinity (48 nM), whereas affinities of other subunit pairs appeared to be low (∼0.21-3.0 µM). The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis. We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities. No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly. Multiple interactions of variable affinity in the stalk region may be important to the mechanism of reversible dissociation of the intact V-ATPase.

Show MeSH
Related in: MedlinePlus