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Thallmair, V.

Publications and source records attributed to Thallmair, V..

2 recordsLinked to original sources
bioRxiv2020

Local PI(4,5)P2 pool dynamics detected by the coincidence biosensor tubbyCT maintain the integrity of ER-PM junctions during PLC signaling

Phosphoinositides (PIs) are important signaling molecules and determinants of membrane identity in the eukaryotic plasma membrane, where they multi-task in divergent signaling pathways. Signaling pleiotropy likely depends on distinct PI pools in the same membrane, although the physical definition of such pools has remained ambiguous. PI(4,5)P2, specifically, is also the precursor for the second messengers in the Gq/PLC pathway, IP3 and DAG, and is broken down by PLC{beta} during signaling. Endoplasmic reticulum-plasma membrane contact sites (ER-PM junctions) have emerged as central hubs for lipid transport between both membranes, and specifically for PI homeostasis by supplying the PM with phosphatidylinositol. Here we show that the tubby protein, by virtue of its C-terminal tubby-domain, preferentially localizes to ER-PM junctions by binding to both PI(4,5)P2 and the ER-PM tether E-Syt3. Under conditions of vigorous PI(4,5)P2 consumption by PLC{beta}, additional recruitment of tubby revealed an increase of a local PI(4,5)P2 pool fed by local synthesis through PI kinases. Inhibition of this pool-filling process led to the release of the ER-PM tethers, E-Syts, from the membrane and hence to loss of integrity of the ER-PM contact sites. We conclude that spatiotemporal metabolic channeling of PI synthesis initiated by non-vesicular transport in the ER-PM junctions specifies a local pool of PI(4,5)P2 that is pivotal for the maintenance of homeostatic functions during global depletion of PI(4,5)P2. The findings further suggest that the tubby-like proteins (TULPs), so far known to impact on energy homeostasis and obesity through primary cilia signaling, have an additional function at ER-PM junctions. HIGHLIGHTSO_LIThe tubby domain preferentially assembles into ER-PM junctions due to coincidence detection of PI(4,5)P2 and E-Syt3 C_LIO_LITubby recruitment reveals an increase of a local pool of PI(4,5)P2 in ER-PM junctions during PLC{beta} signaling C_LIO_LIJunctional PI(4,5)P2 dynamics require local synthesis of PI(4,5)P2 C_LIO_LILocal PI(4,5)P2 supply is required for integrity of ER-PM junctions during PLC{beta} activity. C_LI

cell biology↗
bioRxiv2020

A second PI(4,5)P2 binding site determines PI(4,5)P2 sensitivity of the tubby domain

Phosphosinositides (PIs) are lipid signaling molecules that operate by recruiting proteins to cellular membranes via PI recognition domains. Such domains are also used widely as fluorescence-coupled biosensors for cellular PIs. For PI(4,5)P2, the dominant PI of the plasma membrane (PM), only two recognition domains have been characterized in detail and used as sensors. One of them, the tubby domain, which is conserved in the tubby-like protein (TULP) family, is essential for targeting proteins into cilia in a process involving reversible membrane association. However, the PI(4,5)P2 binding properties of tubby domains have remained enigmatic. Here we used coarse-grained molecular dynamics (MD) simulations to explore PI(4,5)P2 binding by the prototypic tubby domain (tubbyCT). While the MD simulations showed a comparatively low PI(4,5)P2 affinity of the previously described canonical binding site, they unexpectedly revealed an adjacent second binding site, consisting of a conserved cationic cluster at the protein-membrane interface. Population of this second site dramatically increased membrane association of tubbyCT. Although less specific than the canonical binding pocket, this second site preferred binding of PI(4,5)P2 over PI(4)P and phosphatidyl serine. Mutations in this site impaired PI(4,5)P2-dependent PM localization in living cells and PI(4,5)P2 interaction in silico. Thus, the second binding site essentially contributes to the effective affinity and hence PM association of the tubby domain. The two-ligand binding mode may serve to sharpen the membrane association-dissociation cycle of TULPs that underlies delivery of ciliary cargo.

biophysics↗