TY - JOUR
T1 - Biochemical and Structural Studies of Yeast Vps4 Oligomerization
AU - Gonciarz, Malgorzata D.
AU - Whitby, Frank G.
AU - Eckert, Debra M.
AU - Kieffer, Collin
AU - Heroux, Annie
AU - Sundquist, Wesley I.
AU - Hill, Christopher P.
N1 - Funding Information:
We thank Ian Huggins for help with protein purification, Heidi Schubert for advice with crystallography, and a reviewer for suggesting the assignment of electron density as an ethylene glycol molecule. We gratefully acknowledge the DNA Synthesis and Sequencing Core Facilities at the University of Utah. Data for this study were measured at beamline X25 of the National Synchrotron Light Source. Financial support comes principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy and from the National Center for Research Resources of the National Institutes of Health. This work was supported by National Institutes of Health Grants AI51174 (to W.I.S.) and P50GM082545 (to W.I.S. and C.P.H.).
PY - 2008/12/26
Y1 - 2008/12/26
N2 - The ESCRT (endosomal sorting complexes required for transport) pathway functions in vesicle formation at the multivesicular body, the budding of enveloped RNA viruses such as HIV-1, and the final abscission stage of cytokinesis. As the only known enzyme in the ESCRT pathway, the AAA ATPase (ATPase associated with diverse cellular activities) Vps4 provides the energy required for multiple rounds of vesicle formation. Like other Vps4 proteins, yeast Vps4 cycles through two states: a catalytically inactive disassembled state that we show here is a dimer and a catalytically active higher-order assembly that we have modeled as a dodecamer composed of two stacked hexameric rings. We also report crystal structures of yeast Vps4 proteins in the apo- and ATPγS [adenosine 5′-O-(3-thiotriphosphate)]-bound states. In both cases, Vps4 subunits assembled into continuous helices with 6-fold screw axes that are analogous to helices seen previously in other Vps4 crystal forms. The helices are stabilized by extensive interactions between the large and small AAA ATPase domains of adjacent Vps4 subunits, suggesting that these contact surfaces may be used to build both the catalytically active dodecamer and catalytically inactive dimer. Consistent with this model, we have identified interface mutants that specifically inhibit Vps4 dimerization, dodecamerization, or both. Thus, the Vps4 dimer and dodecamer likely form distinct but overlapping interfaces. Finally, our structural studies have allowed us to model the conformation of a conserved loop (pore loop 2) that is predicted to form an arginine-rich pore at the center of one of the Vps4 hexameric rings. Our mutational analyses demonstrate that pore loop 2 residues Arg241 and Arg251 are required for efficient HIV-1 budding, thereby supporting a role for this "arginine collar" in Vps4 function.
AB - The ESCRT (endosomal sorting complexes required for transport) pathway functions in vesicle formation at the multivesicular body, the budding of enveloped RNA viruses such as HIV-1, and the final abscission stage of cytokinesis. As the only known enzyme in the ESCRT pathway, the AAA ATPase (ATPase associated with diverse cellular activities) Vps4 provides the energy required for multiple rounds of vesicle formation. Like other Vps4 proteins, yeast Vps4 cycles through two states: a catalytically inactive disassembled state that we show here is a dimer and a catalytically active higher-order assembly that we have modeled as a dodecamer composed of two stacked hexameric rings. We also report crystal structures of yeast Vps4 proteins in the apo- and ATPγS [adenosine 5′-O-(3-thiotriphosphate)]-bound states. In both cases, Vps4 subunits assembled into continuous helices with 6-fold screw axes that are analogous to helices seen previously in other Vps4 crystal forms. The helices are stabilized by extensive interactions between the large and small AAA ATPase domains of adjacent Vps4 subunits, suggesting that these contact surfaces may be used to build both the catalytically active dodecamer and catalytically inactive dimer. Consistent with this model, we have identified interface mutants that specifically inhibit Vps4 dimerization, dodecamerization, or both. Thus, the Vps4 dimer and dodecamer likely form distinct but overlapping interfaces. Finally, our structural studies have allowed us to model the conformation of a conserved loop (pore loop 2) that is predicted to form an arginine-rich pore at the center of one of the Vps4 hexameric rings. Our mutational analyses demonstrate that pore loop 2 residues Arg241 and Arg251 are required for efficient HIV-1 budding, thereby supporting a role for this "arginine collar" in Vps4 function.
KW - AAA ATPase
KW - Vps4
KW - X-ray crystallography
KW - multivesicular body pathway
KW - oligomerization
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U2 - 10.1016/j.jmb.2008.09.066
DO - 10.1016/j.jmb.2008.09.066
M3 - Article
C2 - 18929572
AN - SCOPUS:56249119525
VL - 384
SP - 878
EP - 895
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 4
ER -