Phosphoinositide Messengers in Cellular Signaling and Trafficking

Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are important for many functions in all eukaryotic cells. PI(4,5)P₂, for example, is a critical regulatory lipid found in the plasma membrane (PM) that controls the activity of many ion channels and transporters, in addition to being a precursor of the second messengers inositol 1,4,5-trisphosphate (InsP₃) and diacylglycerol (DAG). PI4P, on the other hand, plays and important role in the Golgi, while PI3P controls endosomal vesicle dynamics and autophagy. How PI, which is synthesized in the endoplasmic reticulum (ER), reaches membranes where PPIns form and whose function is poorly understood. PI–transfer proteins (PITPs) have been identified and studied extensively, but direct proof that PITPs are indeed responsible for delivering PI to various organellar membranes has been lacking. This is partially the result of functional redundancies between the various PITPs that are found in higher organisms. Mammalian PITPs belong to one of two classes: Class I PITPs are small proteins (about 30 kDa) encoded by two separate genes, PITPNA and PITPNB, with the latter producing two splice forms that differ at their C-termini. Class II PITPs, such as Nir2 and Nir3 (or PITPNM1 and PITPNM2), are larger proteins that also have a canonical PITP domain at their N-termini, which is analogous to the Class I PITP fold and is followed by several additional domains that mediate interactions with membranes as well as other proteins. In this study, we used a recently identified inhibitor, VT01454, that specifically targets Class I but not Class II PITPs, to unravel the roles of Class I PITPs in lipid metabolism and cellular signaling. Our collaborators in Evžen Boura’s group solved the structure of the inhibitor-bound PITPNA to gain insight into the mode of inhibition. In functional studies we found that Class I PITPs not only distribute PI for PPIns production in various organelles, including the PM and late endosome/lysosomes, but their inhibition significantly reduced the levels of other lipids, including phosphatidylserine, di- and triacylglycerols, and caused prominent increases in phosphatidic acid. In contrast, and contrary to expectations, VT01454 did not inhibit Golgi PI4P formation or resting PM PI(4,5)P₂ levels. However, the recovery of the PM pool of PI(4,5)P₂ after receptor-mediated hydrolysis required both Class I and Class II PITPs. Overall, the studies showed that Class I PITPs are important to supply membranes with PI to generate the PPIn pools, but that their acute inhibition also has a major impact on the overall cellular lipid landscape. The importance of these studies is that they help us better understand how cells synthesize and distribute their lipids to form the various membranes and how alterations in these processes can lead to lipid-storage disorders, including lipodystrophies or obesity.

Phosphatidylserine (PS) is an important anionic phospholipid that is synthesized within the ER. While PS shows highest enrichment and serves important functional roles in the PM, its role in the nucleus is poorly explored. In these experiments, we used established PS biosensors targeted to the nucleus and found that PS is also uniquely enriched in the inner nuclear membrane (INM) and the nuclear reticulum (NR). Assessment of the PS density in the different leaflets of the endoplasmic reticulum (ER) and the nuclear envelope, showed that the PS levels are highest in the INM, although they are not as high as they are at the PM. In search of proteins that are affected by PS in the nucleus, we studied CCTa and Lipin1a, two key enzymes important for phosphatidylcholine (PC) biosynthesis. The enzymes reside in the nuclear matrix in resting cells but rapidly translocate to the INM and NR in response to oleic acid treatment even before they move to the cytoplasm to associate with lipid droplets (LDs). Using an enzymatic strategy to convert PS to phosphatidylethanolamine (PE) to deplete nuclear PS levels, we found that the membrane translocation response of both CCTa and Lipin1a required the presence of PS in the nuclear membranes. We also identified the PS–interacting regions within the M-domain of CCTa and the M-Lip domain of Lipin1a and also found that lipid droplet formation is altered by manipulations of nuclear PS availability. Our studies revealed an unrecognized regulatory role of nuclear PS levels in the control of key PC–synthesizing enzymes within the nucleus. The importance of these studies is that they identified a critical new element in the regulatory sequence by which cells respond to fatty acid exposure, a process that is important in the control of fat storage and mobilization.

Mitochondria exist as dynamic networks that undergo coordinated cycles of fusion and fission, processes that are critical for the proper function of the organelle and, hence, are essential for many cellular functions such as metabolism, signaling, and organelle quality control. The proteins that serve as determinants of the mitochondrial network architecture have been well characterized. Specifically, the process of mitochondrial fission is executed by dynamin-related protein 1 (Drp1), a conserved dynamin-like GTPase, which works with outer mitochondrial membrane (OMM)–localized adaptors, and is influenced by inter-organelle contacts, and cytoskeletal interactions to orchestrate mitochondrial division. Mitochondrial membrane–fusion events are similarly controlled by specific families of dynamin-like GTPases, with two structurally related transmembrane mitofusins (Mfn1/2)10 functioning to facilitate OMM fusion. Defects in these processes have been identified as underlying causes in a variety of human diseases. While much is known about the control of protein assemblies that regulate mitochondrial dynamics, the influence of specific membrane lipids in the mitochondrial membranes on the mitochondrial fusion and fission processes remains poorly understood. In a series of studies, we developed and employed new molecular tools to evaluate the direct effects of membrane lipid composition, and diacylglycerol (DAG) in particular, on mitochondrial dynamics. We designed engineered lipid-modifying enzymes that could be acutely driven to the cytosolic leaflet of the OMM to selectively convert phosphatidylinositol (PI) to DAG in those membranes. We found that localized production of DAG initiates rapid and uniform fragmentation of the mitochondrial network and that this effect depends on the presence of Drp1. We also showed that acute DAG production also recruits the Bin/Amphiphysin/Rvs (BAR) domain–containing proteins endophilin B1 and B2 (EndoB1/2) to the mitochondrial surface, inducing acute OMM deformation. In collaboration with Thomas Pucadyil’s group, we found that the presence of DAG directly facilitates the membrane binding and self-assembly of Drp1 using in vitro reconstitution experiments. In addition, the presence of DAG in these lipid templates recruited both isoforms of EndoB, causing massive membrane tubulations. As we found no direct interaction between EndoB1/2 and Drp1, we concluded that localized DAG production activates multiple parallel pathways in the OMM for membrane remodeling. Taken together, these experiments showed that OMM lipid composition directly affects mitochondrial morphology, including activation of Drp1–dependent fission events.