Activation of human VPS4A by ESCRT-III proteins reveals ability of substrates to relieve enzyme autoinhibition

Samuel Merrill, Phyllis I. Hanson

Research output: Contribution to journalArticle

Abstract

VPS4 proteins are AAA+ ATPases required to form multive-sicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ∼50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA+ ATPases, but this has been difficult to directly demonstrate. We found that concentrating His 6-VPS4A on liposomes containing Ni2+-nitrilotriacetic acid-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers.

Original languageEnglish (US)
Pages (from-to)35428-35438
Number of pages11
JournalJournal of Biological Chemistry
Volume285
Issue number46
DOIs
StatePublished - Nov 12 2010
Externally publishedYes

Fingerprint

Endosomal Sorting Complexes Required for Transport
Chemical activation
Substrates
Enzymes
Proteins
Oligomers
Adenosine Triphosphatases
His-His-His-His-His-His
Liposomes
Adenosine Triphosphate
Nitrilotriacetic Acid
Cytokinesis
Virion
Amino Acid Sequence
Hydrolysis
Polymers
Membranes
Lipids
Amino Acids

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Activation of human VPS4A by ESCRT-III proteins reveals ability of substrates to relieve enzyme autoinhibition. / Merrill, Samuel; Hanson, Phyllis I.

In: Journal of Biological Chemistry, Vol. 285, No. 46, 12.11.2010, p. 35428-35438.

Research output: Contribution to journalArticle

@article{c43257023e9348e3b54c647df8874d55,
title = "Activation of human VPS4A by ESCRT-III proteins reveals ability of substrates to relieve enzyme autoinhibition",
abstract = "VPS4 proteins are AAA+ ATPases required to form multive-sicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ∼50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA+ ATPases, but this has been difficult to directly demonstrate. We found that concentrating His 6-VPS4A on liposomes containing Ni2+-nitrilotriacetic acid-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers.",
author = "Samuel Merrill and Hanson, {Phyllis I.}",
year = "2010",
month = "11",
day = "12",
doi = "10.1074/jbc.M110.126318",
language = "English (US)",
volume = "285",
pages = "35428--35438",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "46",

}

TY - JOUR

T1 - Activation of human VPS4A by ESCRT-III proteins reveals ability of substrates to relieve enzyme autoinhibition

AU - Merrill, Samuel

AU - Hanson, Phyllis I.

PY - 2010/11/12

Y1 - 2010/11/12

N2 - VPS4 proteins are AAA+ ATPases required to form multive-sicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ∼50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA+ ATPases, but this has been difficult to directly demonstrate. We found that concentrating His 6-VPS4A on liposomes containing Ni2+-nitrilotriacetic acid-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers.

AB - VPS4 proteins are AAA+ ATPases required to form multive-sicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ∼50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA+ ATPases, but this has been difficult to directly demonstrate. We found that concentrating His 6-VPS4A on liposomes containing Ni2+-nitrilotriacetic acid-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers.

UR - http://www.scopus.com/inward/record.url?scp=78149276258&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78149276258&partnerID=8YFLogxK

U2 - 10.1074/jbc.M110.126318

DO - 10.1074/jbc.M110.126318

M3 - Article

C2 - 20805225

AN - SCOPUS:78149276258

VL - 285

SP - 35428

EP - 35438

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 46

ER -