Structural and functional characterization of the dominant negative P‐loop lysine mutation in the dynamin superfamily protein Vps1

Abstract

Dynamin-superfamily proteins (DSPs) are large self-assembling mechanochemical GTPases that harness GTP hydrolysis to drive membrane remodeling events needed for many cellular processes. Mutation to alanine of a fully conserved lysine within the P-loop of the DSP GTPase domain results in abrogation of GTPase activity. This mutant has been widely used in the context of several DSPs as a dominant-negative to impair DSP-dependent processes.However, the precise deficit of the P-loop K to A mutation remains an open question. Here, we use biophysical, biochemical and structural approaches to characterize this mutant in the context of the endosomal DSP Vps1. We show that the Vps1 P-loop K to A mutant binds nucleotide with an affinity similar to wild type but exhibits defects in the organization of the GTPase active site that explain the lack of hydrolysis. In cells, Vps1 and Dnm1 bearing the P-loop K to A mutation are defective in disassembly. These mutants become trapped in assemblies at the typical site of action of the DSP. This work provides mechanistic insight into the widely-used DSP P-loop K to A mutation and the basis of its dominant-negative effects in the cell.

The dynamin-related protein Vps1 and the peroxisomal membrane protein Pex27 function together during peroxisome fission

“…3 A,B). Vps1-K42A–GFP is mainly localised to endosomal structures ( Tornabene et al, 2020 ; Varlakhanova et al, 2018 ), but we also observed colocalisation of the GFP signal with peroxisomes ( Fig. 3 B).…”

“…The lack of Vps1–GFP localisation to peroxisomes might be a consequence of Vps1 being present briefly during a fission event as has been reported for the scission of endocytic vesicles from the plasma membrane (<10 s) ( Smaczynska-de Rooji et al, 2010 ). To visualise Vps1 on peroxisomes, we used a GTPase-defective mutant (Vps1-K42A) that locks the protein in a constricted helical assembly on its membrane substrate ( Sundborger et al, 2014 ; Tornabene et al, 2020 ; Varlakhanova et al, 2018 ). This mutant does not restore peroxisome fission in vps1Δ/dnm1Δ cells ( Fig.…”

A PX-BAR protein Mvp1/SNX8 and a dynamin-like GTPase Vps1 drive endosomal recycling

“…In this mutant, membrane scission of clathrin-coated vesicles is blocked. This lysine residue is widely conserved in dynamin-related GTPases, including Vps1 ( Figure 4F , Figure 4—figure supplement 1E ; Varlakhanova et al, 2018 ; Tornabene et al, 2020 ). To test if the GTP hydrolysis activity of Vps1 is required for Vps55 recycling, we examined Vps55 localization in vps1 K42A mutants.…”

A PX-BAR protein Mvp1/SNX8 and a dynamin-like GTPase Vps1 drive endosomal recycling

“…In this mutant, membrane scission of clathrin-coated vesicles is blocked. This lysine residue is widely conserved in dynamin-related GTPases, including Vps1 (Figure 4F and S4E; Varlakhanova et al, 2018; Tornabene et al, 2020). To test if the GTP hydrolysis activity of Vps1 is required for Vps55 recycling, we examined Vps55 localization in vps1 K42A mutants.…”