Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan...

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Main Authors:	Guo, Zhong, Johnston, Wayne, Kovtun, Oleksiy, Mureev, Sergey, Bröcker, Cornelia, Ungermann, Christian, Alexandrov, Kirill
Format:	Online
Language:	English
Published:	Public Library of Science 2013
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3846719/

id	pubmed-3846719
recordtype	oai_dc
spelling	pubmed-38467192013-12-05 Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes Guo, Zhong Johnston, Wayne Kovtun, Oleksiy Mureev, Sergey Bröcker, Cornelia Ungermann, Christian Alexandrov, Kirill Research Article Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data. Public Library of Science 2013-12-02 /pmc/articles/PMC3846719/ /pubmed/24312556 http://dx.doi.org/10.1371/journal.pone.0081534 Text en © 2013 Guo et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Guo, Zhong Johnston, Wayne Kovtun, Oleksiy Mureev, Sergey Bröcker, Cornelia Ungermann, Christian Alexandrov, Kirill
spellingShingle	Guo, Zhong Johnston, Wayne Kovtun, Oleksiy Mureev, Sergey Bröcker, Cornelia Ungermann, Christian Alexandrov, Kirill Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes
author_facet	Guo, Zhong Johnston, Wayne Kovtun, Oleksiy Mureev, Sergey Bröcker, Cornelia Ungermann, Christian Alexandrov, Kirill
author_sort	Guo, Zhong
title	Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes
title_short	Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes
title_full	Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes
title_fullStr	Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes
title_full_unstemmed	Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes
title_sort	subunit organisation of in vitro reconstituted hops and corvet multisubunit membrane tethering complexes
description	Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data.
publisher	Public Library of Science
publishDate	2013
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3846719/
_version_	1612032683592908800

Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

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