Specific changes in phospholipid content are a hallmark of the membranes of maturing endosomes and phagosomes, but is it unclear how this is controlled. Naufer et al. now show that acidification of the lumen of endosomes and phagosomes triggers dissociation of the Vps34 lipid kinase from these organelles, which terminates PtdIns(3)P synthesis and signaling.

Phosphatidic acid (PA) and diacylglycerol (DAG) are key signaling molecules and important precursors for the biosynthesis of all glycerolipids found in eukaryotes. Research conducted in the model organism Saccharomyces cerevisiae has been at the forefront of the identification of the enzymes involved in the metabolism and transport of PA and DAG. Both these lipids can alter the local physical properties of membranes by introducing negative curvature, but the anionic nature of the phosphomonoester headgroup in PA sets it apart from DAG. As a result, the mechanisms underlying PA and DAG interaction with other lipids and proteins are notoriously different. This is apparent from the analysis of the protein domains responsible for recognition and binding to each of these lipids. We review the current evidence obtained using the PA-binding proteins and domains fused to fluorescent proteins for in vivo tracking of PA pools in yeast. In addition, we present original results for visualization of DAG pools in yeast using the C1 domain from mammalian PKCδ. An emerging first cellular map of the distribution of PA and DAG pools in actively growing yeast is discussed.

Diacylglycerol (DAG) is a key signaling lipid and intermediate in lipid metabolism. Our knowledge of DAG distribution and dynamics in cell membranes is limited. Using live‐cell fluorescence microscopy we investigated the localization of yeast cytosolic‐facing pools of DAG in response to conditions where lipid homeostasis and DAG levels were known to be altered. Two main pools were monitored over time using DAG sensors. One pool was associated with vacuolar membranes and the other localized to sites of polarized growth. Dynamic changes in DAG distribution were observed during resumption of growth from stationary phase, when DAG is used to support phospholipid synthesis for membrane proliferation. Vacuolar membranes experienced constant morphological changes displaying DAG enriched microdomains coexisting with liquid‐disordered areas demarcated by Vph1. Formation of these domains was dependent on triacylglycerol (TAG) lipolysis. DAG domains and puncta were closely connected to lipid droplets. Lack of conversion of DAG to phosphatidate in growth conditions dependent on TAG mobilization, led to the accumulation of DAG in a vacuolar‐associated compartment, impacting the polarized distribution of DAG at budding sites. DAG polarization was also regulated by phosphatidylserine synthesis/traffic and sphingolipid synthesis in the Golgi.

Actively maintained close appositions between organelle membranes, also known as contact sites, enable the efficient transfer of biomolecules between cellular compartments. Several such sites have been described as well as their tethering machineries. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize contact site proteomes, we established a high-throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, several of which are poorly characterized, on the background of 1165 strains expressing a mCherry-tagged yeast protein that has a cellular punctate distribution (a hallmark of contact sites), under regulation of the strong TEF2 promoter. By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified three homologs of Vps13 and Atg2 that are residents of multiple contact sites. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell. Overall, our analysis expands the universe of contact site residents and effectors and creates a rich database to mine for new functions, tethers, and regulators.

Fundamental changes in the composition and distribution of lipids within the brain are believed to contribute to the cognitive decline associated with Alzheimer’s disease (AD). The mechanisms by which these changes in lipid composition affect cellular function and ultimately cognition are not well understood. Although “candidate gene” approaches can provide insight into the effects of dysregulated lipid metabolism they require a preexisting understanding of the molecular targets of individual lipid species. In this report we combine unbiased gene expression profiling with a genome-wide chemogenomic screen to identify the mitochondria as an important downstream target of PC(O-16:0/2:0), a neurotoxic lipid species elevated in AD. Further examination revealed that PC(O-16:0/2:0) similarly promotes a global increase in ceramide accumulation in human neurons which was associated with mitochondrial-derived reactive oxygen species (ROS) and toxicity. These findings suggest that PC(O-16:0/2:0)-dependent mitochondrial dysfunction may be an underlying contributing factor to the ROS production associated with AD.