Biology subjects

Starr, M. L.

Publications and source records attributed to Starr, M. L..

4 recordsLinked to original sources

bioRxiv2019

Copper Blocks V-ATPase Activity and SNARE Complex Formation to Inhibit Yeast Vacuole Fusion

The accumulation of Copper in organisms can lead to altered functions of various pathways, and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg+, Cd2+, and Cu2+ are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations. Excess copper ions can subsequently be transported into the vacuole lumen by an unknown mechanism. Transport across membranes requires the reduction of Cu2+ to Cu+. Labile copper ions can interact with membranes to alter fluidity, lateral phase separation and fusion. Here we found that CuCl2 potently inhibited vacuole fusion by blocking SNARE pairing. This was accompanied by the inhibition of V-ATPase H+ pumping. Deletion of the vacuolar reductase Fre6 had no effect on the inhibition of fusion by copper. This suggests that that Cu2+ is responsible for the inhibition of vacuole fusion and V-ATPase function. This notion is supported by the differential effects chelators. The Cu2+-specific chelator TETA rescued fusion, whereas the Cu+-specific chelator BCS had no effect on the inhibited fusion.

biochemistry↗

bioRxiv2019

Phosphatidylinositol 3,5-Bisphosphate regulates Ca2+ Transport During Yeast Vacuolar Fusion through Activation of the Ca2+ ATPase Pmc1

The transport of Ca2+ across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles during hyperosmotic shock become fragmented through fission events that require Ca2+ efflux of their luminal stores through the TRP channel Yvc1. This requires the production of the lipid PI(3,5)P2 by Fab1. Ca2+ is also released during vacuole fusion upon trans-SNARE complex assembly, however, the role of PI(3,5)P2 remains unclear. Here we demonstrate that elevated PI(3,5)P2 levels abolish Ca2+ efflux during fusion, indicating that PI(3,5)P2 has opposing effects on Ca2+ transport in fission versus fusion. Notably, Ca2+ efflux was enhanced when PI(3,5)P2 levels were reduced. Importantly, the effect of PI(3,5)P2 on Ca2+ flux was independent of Yvc1. Rather, the effect was dependent on the Ca2+ pump Pmc1. Vacuoles lacking Pmc1 were resistant to the effects of PI(3,5)P2, while those lacking Yvc1 remained sensitive. Furthermore altering PI(3,5)P2 levels affects the interactions of Pmc1 with the Vo component Vph1 and the R-SNARE Nyv1. We now propose a model in which elevated PI(3,5)P2 activates continued Pmc1 function to prevent the accumulation of released extraluminal Ca2+. SummaryDuring osmotic stress PI(3,5)P2 triggers Ca2+ release from vacuoles. Here we show PI(3,5)P2 stimulates Ca2+ uptake by vacuoles during fusion, illustrating that it has a dual role in Ca2+ transport.

biochemistry↗

bioRxiv2018

Phosphatidylinositol 3,5-Bisphosphate Regulates Yeast Vacuole Fusion at the Transition between trans-SNARE Complex Formation and Hemifusion

Phosphoinositides (PIs) regulate myriad cellular functions including membrane fusion, as exemplified by the yeast vacuole, which uses various PIs at different stages of fusion. In light of this, the effect of phos-phatidylinositol 3,5-bisphosphate [PI(3,5)P2] on vacuole fusion remains unknown. PI(3,5)P2 is made by the PI3P 5-kinase Fab1/PIKfyve and has been characterized as a regulator of vacuole fission during hyperosmotic shock where it interacts with the TRP family Ca2+ channel Yvc1. Here we demonstrate that exogenously added dioctanoyl (C8) PI(3,5)P2 abolishes homotypic vacuole fusion. This effect was not linked to interactions with Yvc1, as fusion was equally affected using yvc1{Delta} vacuoles. Thus, the effects of C8-PI(3,5)P2 on fusion versus fission operate through distinct mechanisms. Further testing showed that C8-PI(3,5)P2 inhibited vacuole fusion after the formation of trans-SNARE pairs. Although SNARE complex formation was unaffected we found that C8-PI(3,5)P2 strongly inhibited hemifusion. Overproduction of endogenous PI(3,5)P2 by the fab1T2250A hyperactive kinase mutant also inhibited at the hemifusion stage, bolstering the model in which PI(3,5)P2 inhibits fusion when present elevated levels. Taken together, this work identifies a novel function for PI(3,5)P2 as a negative regulator of vacuolar fusion. Moreover, it suggests that this lipid acts as a molecular switch between fission and fusion.

biochemistry↗

bioRxiv2018

Phosphatidic acid inhibits SNARE priming by inducing conformational changes in Sec18 protomers

Eukaryotic homeostasis relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are re-activated by Sec18/NSF driven disassembly to enable a new round of fusion. We previously found that phosphatidic acid (PA) binds Sec18 to sequester it from SNAREs. Dephosphorylation of PA dissociates Sec18 from the membrane allowing it to engage SNARE complexes. We now report that PA induces conformational changes in Sec18 protomers, while hexameric Sec18 cannot bind PA membranes. The association of Sec18 with PA was shown to be sensitive to membrane curvature, suggesting that regulation could vary on different organelles in a curvature dependent manner. Molecular dynamics showed that PA binding sites exist on the D1 and D2 domains of Sec18 and that residues needed for binding were masked in the hexameric form of the protein. Together these data indicate that PA regulates Sec18 function through altering protein architecture and stabilizing membrane-bound protomers.

biochemistry↗