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

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.

Structural Identification of the Vps18 β-Propeller Reveals a Critical Role in the HOPS Complex Stability and Function

“…We previously showed that CORVET assembly occurs in the absence of the N-terminal domains of CORVET Vps3 and Vps8, whereas the C-terminal domains are critical for assembly (32). In agreement with this, the in vitro assembly of the C-terminal predicted ␣-helical segments resulted in a HOPS core complex (33), suggesting that the S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P Vps18 NTD -GST was incubated with the indicated Vps11-Vps39 dimer, the Vps11-Vps39-Vps18 trimer, with the Vps16-Vps33 dimer, and with the entire HOPS complex carrying a GFP tag on Vps33. The GST pulldown was conducted as before (14).…”

Maintaining order: COG complex controls Golgi trafficking, processing, and sorting

“…This model is supported by in vitro reconstitution experiments [52,72,136] in which purified assembled COG complex showed twofold stimulation of vesicle fusion reaction. It is also in agreement with recent models proposed for the HOPS tethering complex [137,138], but fails to explain the existence of membrane-attached COG subcomplexes [53,139] and the dispensability of lobe B subunits in yeast cells.…”

Diversification of CORVET tethers facilitates transport complexity inTetrahymena thermophila

“…However, we note that such hybrid CORVET/HOPS tethers have to date only been detected in yeast, and under conditions in which subunit overexpression could potentially result in non-physiological complexes. Vps8 paralogs is consistent with genetic, biochemical and structural mapping of subunit interactions (Guo et al, 2013;Hunter et al, 2017;Plemel et al, 2011) (Brocker et al, 2012Chou et al, 2016).…”