Increased Phospholipid Flux Bypasses Overlapping Essential Requirements for the Yeast Sac1p Phosphoinositide Phosphatase and ER-PM Membrane Contact Sites.

In budding yeast cells, much of the inner surface of the plasma membrane (PM) is covered with the endoplasmic reticulum (ER). This association is mediated by seven ER membrane proteins that confer cortical ER-PM association at membrane contact sites (MCSs). Several of these membrane "tether" proteins are known to physically interact with the phosphoinositide phosphatase Sac1p. However, it is unclear how or if these interactions are necessary for their interdependent functions. We find that SAC1 inactivation in cells lacking the homologous synaptojanin-like genes INP52 and INP53 results in a significant increase in cortical ER-PM MCSs. We show in sac1Δ, sac1tsinp52Δ inp53Δ, or Δ-super-tether (Δ-s-tether) cells lacking all seven ER-PM tethering genes that phospholipid biosynthesis is disrupted and phosphoinositide distribution is altered. Furthermore, SAC1 deletion in Δ-s-tether cells results in lethality, indicating a functional overlap between SAC1 and ER-PM tethering genes. Transcriptomic profiling indicates that SAC1 inactivation in either Δ-s-tether or inp52Δ inp53Δ cells induces an ER membrane stress response and elicits phosphoinositide-dependent changes in expression of autophagy genes. In addition, by isolating high-copy suppressors that rescue sac1Δ Δ-s-tether lethality, we find that key phospholipid biosynthesis genes bypass the overlapping function of SAC1 and ER-PM tethers and that overexpression of the phosphatidylserine/phosphatidylinositol-4-phosphate transfer protein Osh6 also provides limited suppression. Combined with lipidomic analysis and determinations of intracellular phospholipid distributions, these results suggest that Sac1p and ER phospholipid flux controls lipid distribution to drive Osh6p-dependent phosphatidylserine/phosphatidylinositol-4-phosphate counter-exchange at ER-PM MCSs.

ER-PM membrane contact site regulation by yeast ORPs and membrane stress pathways.

In yeast, at least seven proteins (Ice2p, Ist2p, Scs2/22p, Tcb1-Tcb3p) affect cortical endoplasmic reticulum (ER) tethering and contact with the plasma membrane (PM). In Δ-super-tether (Δ-s-tether) cells that lack these tethers, cortical ER-PM association is all but gone. Yeast OSBP homologue (Osh) proteins are also implicated in membrane contact site (MCS) assembly, perhaps as subunits for multicomponent tethers, though their function at MCSs involves intermembrane lipid transfer. Paradoxically, when analyzed by fluorescence and electron microscopy, the elimination of the OSH gene family does not reduce cortical ER-PM association but dramatically increases it. In response to the inactivation of all Osh proteins, the yeast E-Syt (extended-synaptotagmin) homologue Tcb3p is post-transcriptionally upregulated thereby generating additional Tcb3p-dependent ER-PM MCSs for recruiting more cortical ER to the PM. Although the elimination of OSH genes and the deletion of ER-PM tether genes have divergent effects on cortical ER-PM association, both elicit the Environmental Stress Response (ESR). Through comparisons of transcriptomic profiles of cells lacking OSH genes or ER-PM tethers, changes in ESR expression are partially manifested through the induction of the HOG (high-osmolarity glycerol) PM stress pathway or the ER-specific UPR (unfolded protein response) pathway, respectively. Defects in either UPR or HOG pathways also increase ER-PM MCSs, and expression of extra "artificial ER-PM membrane staples" rescues growth of UPR mutants challenged with lethal ER stress. Transcriptome analysis of OSH and Δ-s-tether mutants also revealed dysregulation of inositol-dependent phospholipid gene expression, and the combined lethality of osh4Δ and Δ-s-tether mutations is suppressed by overexpression of the phosphatidic acid biosynthetic gene, DGK1. These findings establish that the Tcb3p tether is induced by ER and PM stresses and ER-PM MCSs augment responses to membrane stresses, which are integrated through the broader ESR pathway.

Sticking With It: ER-PM Membrane Contact Sites as a Coordinating Nexus for Regulating Lipids and Proteins at the Cell Cortex.

Membrane contact sites between the cortical endoplasmic reticulum (ER) and the plasma membrane (PM) provide a direct conduit for small molecule transfer and signaling between the two largest membranes of the cell. Contact is established through ER integral membrane proteins that physically tether the two membranes together, though the general mechanism is remarkably non-specific given the diversity of different tethering proteins. Primary tethers including VAMP-associated proteins (VAPs), Anoctamin/TMEM16/Ist2p homologs, and extended synaptotagmins (E-Syts), are largely conserved in most eukaryotes and are both necessary and sufficient for establishing ER-PM association. In addition, other species-specific ER-PM tether proteins impart unique functional attributes to both membranes at the cell cortex. This review distils recent functional and structural findings about conserved and species-specific tethers that form ER-PM contact sites, with an emphasis on their roles in the coordinate regulation of lipid metabolism, cellular structure, and responses to membrane stress.

Substrate-decoupled, bulk-acoustic wave gyroscopes: Design and evaluation of next-generation environmentally robust devices.

This paper reports on a new type of high-frequency mode-matched gyroscope with significantly reduced dependencies on environmental stimuli such as temperature, vibration, and shock. A novel stress-isolation system is used to effectively decouple an axis-symmetric bulk-acoustic wave (BAW) vibratory gyro from its substrate, minimizing the effect that external sources of error have on the offset and scale factor of the device. Substrate-decoupled (SD) BAW gyros with a resonance frequency of 4.3 MHz and Q values near 60 000 were implemented using the high aspect ratio poly and single-crystal silicon (HARPSS) process to achieve ultra-narrow capacitive gaps. Wafer-level packaged sensors were interfaced with a customized application-specific integrated circuit (ASIC) to achieve low variations in the offset across temperature (±26° s-1 from -40 to 85 °C), supreme random-vibration immunity (0.012° s-1 gRMS -1) and excellent shock rejection. With a scale factor of 800 µV (°s-1)-1, the SD-BAW gyro system attains a large full-scale range (±1250° s-1) with a non-linearity of less than 0.07%. A measured angle-random walk (ARW) of 0.39°/√h and a bias instability of 10.5°h-1 are dominated by the thermal and flicker noise of the integrated circuit (IC), respectively. Additional measurements using external electronics show bias-instability values as low as 3.5°h-1, which are limited by feed-through signals coupled from the drive loop to the sense channel, which can be further reduced through proper re-routing of the gyroscope pin-out configuration.