Jesse Chao

Phosphatidic acid is a pH biosensor that links membrane biogenesis to metabolism

Intracellular pH and Lipid Metabolism Intracellular pH regulates metabolism by poorly understood mechanisms, but biosensors are likely to be important in this process. Young et al. (p. 1085) took a systems-biology approach in yeast to identify in excess of 200 genes that regulate phospholipid metabolism. They found that the signaling lipid, phosphatidic acid, appeared to act as a cytosolic biosensor via the pH-dependent binding of protein effectors to phosphatidic acid. This pH-dependent mechanism directly affects gene expression and is involved in a pathway in which nutrient availability regulates phospholipid metabolism to control production of membranes. Lipid signaling in yeast is regulated by intracellular pH. Recognition of lipids by proteins is important for their targeting and activation in many signaling pathways, but the mechanisms that regulate such interactions are largely unknown. Here, we found that binding of proteins to the ubiquitous signaling lipid phosphatidic acid (PA) depended on intracellular pH and the protonation state of its phosphate headgroup. In yeast, a rapid decrease in intracellular pH in response to glucose starvation regulated binding of PA to a transcription factor, Opi1, that coordinately repressed phospholipid metabolic genes. This enabled coupling of membrane biogenesis to nutrient availability.

A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria

Tethering of the endoplasmic reticulum to mitochondria by a conserved endoplasmic reticulum complex is needed for the transfer of phospholipids between these organelles.

Plasma membrane—endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis

Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites between the endoplasmic reticulum (ER) and other organelles because many lipid‐synthesizing enzymes are enriched in these contacts. In only a few cases have the enzymes been localized to contacts in vivo and in no instances have the contacts been demonstrated to be required for enzyme function. Here, we show that plasma membrane (PM)—ER contact sites in yeast are required for phosphatidylcholine synthesis and regulate the activity of the phosphatidylethanolamine N‐methyltransferase enzyme, Opi3. Opi3 activity requires Osh3, which localizes to PM–ER contacts where it might facilitate in trans catalysis by Opi3. Thus, membrane contact sites provide a structural mechanism to regulate lipid synthesis.

Polarization of the Endoplasmic Reticulum by ER-Septin Tethering

Polarization of the plasma membrane (PM) into domains is an important mechanism to compartmentalize cellular activities and to establish cell polarity. Polarization requires formation of diffusion barriers that prevent mixing of proteins between domains. Recent studies have uncovered that the endoplasmic reticulum (ER) of budding yeast and neurons is polarized by diffusion barriers, which in neurons controls glutamate signaling in dendritic spines. The molecular identity of these barriers is currently unknown. Here, we show that a direct interaction between the ER protein Scs2 and the septin Shs1 creates the ER diffusion barrier in yeast. Barrier formation requires Epo1, a novel ER-associated subunit of the polarisome that interacts with Scs2 and Shs1. ER-septin tethering polarizes the ER into separate mother and bud domains, one function of which is to position the spindle in the mother until M phase by confining the spindle capture protein Num1 to the mother ER.

Barriers to uniformity within the endoplasmic reticulum.

Differentiating the endoplasmic reticulum (ER) into different physical domains may help the ER spatially regulate its many functions. For example, ER sheets are highly decorated with ribosomes for protein synthesis, whereas tubules usually correspond to smooth ER. Hence, ER morphology may play direct roles in functional diversification within the ER. The ER also makes direct physical contacts with other organelles, called ER junctions, enabling further functional diversification through input from external sources. In yeast, an ER diffusion barrier has now been discovered at the bud neck that compartmentalizes the ER into bud and mother diffusion domains by restricting the lateral diffusion of ER membrane proteins. Therefore, diffusion barriers also likely contribute to functional diversification within the ER by creating suites of molecular factors within ER diffusion domains.

Identification of protein complexes with quantitative proteomics in S. cerevisiae.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach.

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning. Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.