Inositol pyrophosphates activate the vacuolar transport chaperone complex in yeast by disrupting a homotypic SPX domain interaction

Abstract

Many proteins involved in eukaryotic phosphate homeostasis are regulated by SPX domains. In yeast, the vacuolar transporter chaperone (VTC) complex contains two such domains, but mechanistic details of its regulation are not well understood. Here, we show at the atomic level how inositol pyrophosphates interact with SPX domains of subunits Vtc2 and Vtc3 to control the activity of the VTC complex. Vtc2 inhibits the catalytically active VTC subunit Vtc4 by homotypic SPX–SPX interactions via the conserved helix α1 and the previously undescribed helix α7. Binding of inositol pyrophosphates to Vtc2 abrogates this interaction, thus activating the VTC complex. Accordingly, VTC activation is also achieved by site-specific point mutations that disrupt the SPX–SPX interface. Structural data suggest that ligand binding induces reorientation of helix α1 and exposes the modifiable helix α7, which might facilitate its post-translational modification in vivo. The variable composition of these regions within the SPX domain family might contribute to the diversified SPX functions in eukaryotic phosphate homeostasis.

Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81

“…SPX domains interact with or form part of a large variety of proteins that affect P i homeostasis by transporting P i across membranes, converting it into polyphosphates (polyP) or other metabolites, or regulating P i -dependent transcription ( Secco et al, 2012 ). Direct regulation of an SPX-containing protein by synthetic inositol pyrophosphates was shown for the polyP polymerase VTC, the P i transporters Pho91 and the PHR transcription factors in plants ( Gerasimaite et al, 2017 ; Potapenko et al, 2018 ; Wild et al, 2016 ; Ried et al, 2021 ; Wang et al, 2015 ; Liu et al, 2016 ; Dong et al, 2019 ; Liu et al, 2023 ; Pipercevic et al, 2023 ; Guan et al, 2023 ). A firm link suggesting that the SPX domain as the receptor for inositol pyrophosphate regulation was provided by point mutants in the SPX domain that rendered VTC either independent of activation by inositol pyrophosphates or non-responsive to them ( Wild et al, 2016 ).…”

“…However, this binding site discriminates poorly between different inositol pyrophosphates at the level of binding ( Wild et al, 2016 ). By contrast, strong differences are observed in the agonist properties, leading to the suggestion that binding affinity is a poor predictor of inositol pyrophosphate specificity and activity ( Austin and Mayer, 2020 ; Gerasimaite et al, 2017 ; Pipercevic et al, 2023 ).…”

Interaction with IP6K1 supports pyrophosphorylation of substrate proteins by the inositol pyrophosphate 5-IP7

“…Indeed, pyrophosphorylation by 5-IP7 is inhibited in the presence of excess IP6 [10]. A majority of proteins that bind IP6 are also shown to bind IP7 [8,19,35,36]. In most organisms and cell types studied till date, the intracellular abundance of IP6 exceeds that of PP-IPs, although there are some notable exceptions, including the slime mould Dictyostelium discoideum in which IP7 and IP8 levels are similar to those of IP6 [37,38].…”

Inositol pyrophosphate catabolism by three families of phosphatases controls plant growth and development

“…In plants grown under Pi-sufficient conditions, the PP-InsP isomer 1,5-InsP 8 accumulates in cells and binds to SPX (Syg1 Pho81 XPR1) receptor proteins (Dong et al, 2019; Ried et al, 2021; Wild et al, 2016). The ligand-bound receptor undergoes conformational changes (Pipercevic et al, 2023; Wild et al, 2016), for example allowing for the interaction with a family of PHOSPHATE STARVATION RESPONSE (PHR) transcription factors (Rubio et al, 2001; Lv et al, 2014; Puga et al, 2014; Wang et al, 2014; Wild et al, 2016). The coiled-coil oligomerisation and Myb DNA binding domains wrap around the SPX receptor, preventing PHRs from interacting with their target promoters.…”