Abstract
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.
Highlights
Small GTPases of the Rab and Arf families confer specificity and directionality to membrane trafficking steps such as vesicle budding, transport, tethering, docking, and fusion
Exchange of Rab5 for Rab7 is controlled by multiple factors including multisubunit tethering complexes (MTC) CORVET and HOPS that are critical for late endosomal and lysosomal biogenesis [1,2]
Our knowledge of HOPS/CORVET assembly and interactions comes from two-hybrid system analysis, pull down experiments from yeast cells transformed with affinity tagged constructs, or analysis of individual subunits expressed in insect cells [14,15]
Summary
Small GTPases of the Rab and Arf families confer specificity and directionality to membrane trafficking steps such as vesicle budding, transport, tethering, docking, and fusion. HOPS and CORVET remain the least biochemically studied tethering complexes mainly due to the very large size of individual subunits, which preclude expression or reconstitution in prokaryotic expression systems. Our knowledge of HOPS/CORVET assembly and interactions comes from two-hybrid system analysis, pull down experiments from yeast cells transformed with affinity tagged constructs, or analysis of individual subunits expressed in insect cells [14,15].
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