Rewiring phospholipid biosynthesis reveals robustness in membrane homeostasis and uncovers lipid regulatory players.

Intracellular transport of lipids by Lipid Transport Proteins (LTPs) is thought to work alongside vesicular transport to shuttle lipids from their place of synthesis to their destinations. Whereas many LTPs have been identified, it is largely unknown which routes and which LTPs a given lipid utilizes to navigate the multiple membranes of eukaryotic cells. The major and essential phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) can be produced by multiple pathways and, in the case of PE, also at multiple locations. Here, we present an approach in which we simplify and rewire yeast phospholipid synthesis by redirecting PE and PC synthesis reactions to distinct subcellular locations using chimeric enzymes fused to specific organelle targeting motifs. In rewired conditions, viability is expected to depend on homeostatic adaptation to the ensuing lipostatic perturbations and on efficient interorganelle lipid transport. We therefore performed genetic screens to identify factors involved in both of these processes. Among the candidates identified, we find genes linked to transcriptional regulation of lipid homeostasis, lipid metabolism and transport. In particular, we identify a requirement for Csf1 -an uncharacterized protein harboring a Chorein-N lipid transport domain- for survival under certain rewired conditions as well as lipidomic adaptation to cold, implicating Csf1 in interorganelle lipid transport and homeostatic adaptation.

Eugene P. Kennedy’s Legacy: Defining Bacterial Phospholipid Pathways and Function

In the 1950’s and 1960’s Eugene P. Kennedy laid out the blueprint for phospholipid biosynthesis in somatic cells and Escherichia coli, which have been coined the Kennedy Pathways for phospholipid biosynthesis. His research group continued to make seminal contributions in the area of phospholipids until his retirement in the early 1990’s. During these years he mentored many young scientists that continued to build on his early discoveries and who also mentored additional scientists that continue to make important contributions in areas related to phospholipids and membrane biogenesis. This review will focus on the initial E. coli Kennedy Pathways and how his early contributions have laid the foundation for our current understanding of bacterial phospholipid genetics, biochemistry and function as carried on by his scientific progeny and others who have been inspired to study microbial phospholipids.

Mosquito metabolomics reveal that dengue virus replication requires phospholipid reconfiguration via the remodeling cycle

Dengue virus (DENV) subdues cell membranes for its cellular cycle by reconfiguring phospholipids in humans and mosquitoes. Here, we determined how and why DENV reconfigures phospholipids in the mosquito vector. By inhibiting and activating the de novo phospholipid biosynthesis, we demonstrated the antiviral impact of de novo–produced phospholipids. In line with the virus hijacking lipids for its benefit, metabolomics analyses indicated that DENV actively inhibited the de novo phospholipid pathway and instead triggered phospholipid remodeling. We demonstrated the early induction of remodeling during infection by using isotope tracing in mosquito cells. We then confirmed in mosquitoes the antiviral impact of de novo phospholipids by supplementing infectious blood meals with a de novo phospholipid precursor. Eventually, we determined that phospholipid reconfiguration was required for viral genome replication but not for the other steps of the virus cellular cycle. Overall, we now propose that DENV reconfigures phospholipids through the remodeling cycle to modify the endomembrane and facilitate formation of the replication complex. Furthermore, our study identified de novo phospholipid precursor as a blood determinant of DENV human-to-mosquito transmission.

Phospholipid ebb and flow makes mitochondria go

Mitochondria, so much more than just being energy factories, also have the capacity to synthesize macromolecules including phospholipids, particularly cardiolipin (CL) and phosphatidylethanolamine (PE). Phospholipids are vital constituents of mitochondrial membranes, impacting the plethora of functions performed by this organelle. Hence, the orchestrated movement of phospholipids to and from the mitochondrion is essential for cellular integrity. In this review, we capture recent advances in the field of mitochondrial phospholipid biosynthesis and trafficking, highlighting the significance of interorganellar communication, intramitochondrial contact sites, and lipid transfer proteins in maintaining membrane homeostasis. We then discuss the physiological functions of CL and PE, specifically how they associate with protein complexes in mitochondrial membranes to support bioenergetics and maintain mitochondrial architecture.

Recent advances in the understanding of autophagosome biogenesis

Autophagy is a conserved catabolic process critical for cell homeostasis with broad implications for aging and age-associated diseases. A defining feature of autophagy is the de novo formation of a specialized transient organelle, the double-membrane autophagosome. Autophagosomes originate from small vesicular precursors after rapid membrane expansion resulting in the engulfment of a broad spectrum of cytoplasmic cargoes within a few minutes for vacuolar or lysosomal degradation. Recent advances have provided exciting new insights into the molecular mechanisms underlying the assembly of autophagic membranes during autophagosome biogenesis. Specifically, the phospholipid biosynthesis activity of the endoplasmic reticulum and a dedicated membrane-tethering complex between nascent autophagosomes and the endoplasmic reticulum have emerged as key factors in autophagosome formation.

Intracellular Membranes: Conserved Mechanisms of Formation and Regulation Throughout Evolution

Membrane remodeling and phospholipid biosynthesis are normally tightly regulated to maintain the shape and function of cells. Indeed, different physiological mechanisms ensure a precise coordination between de novo phospholipid biosynthesis and modulation of membrane morphology. Interestingly, the overproduction of certain membrane proteins hijack these regulation networks, leading to the formation of impressive intracellular membrane structures in both prokaryotic and eukaryotic cells. The proteins triggering membrane proliferation share two major common features: 1) they promote the formation of highly curved membrane domains and 2) they lead to an enrichment in anionic, cone-shaped phospholipids (cardiolipin or phosphatidic acid) in the newly formed membranes. Taking into account the available examples of membrane proliferation upon protein overproduction, together with the latest biochemical, biophysical and structural data, we explore the relationship between protein synthesis and membrane biogenesis. We propose a mechanism for the formation of these non-physiological intracellular membranes that shares similarities with natural inner membrane structures found in α-proteobacteria, mitochondria and some viruses-infected cells, pointing towards a conserved feature through evolution. We hope that the information discussed in this review will give a better grasp of the biophysical mechanisms behind physiological and induced intracellular membrane proliferation, inspiring new biotechnological applications.

Membrane fluidity adjusts the insertion of the transacylase PlsX to regulate phospholipid biosynthesis in Gram-positive bacteria

PlsX plays a central role in the coordination of fatty acid and phospholipid biosynthesis in Gram-positive bacteria. PlsX is a peripheral membrane acyltransferase that catalyzes the conversion of acyl-ACP to acyl-phosphate, which is in turn utilized by the polytopic membrane acyltransferase PlsY on the pathway of bacterial phospholipid biosynthesis. We have recently studied the interaction between PlsX and membrane phospholipids in vivo and in vitro, and observed that membrane association is necessary for the efficient transfer of acyl-phosphate to PlsY. However, understanding the molecular basis of such a channeling mechanism remains a major challenge. Here, we disentangle the binding and insertion events of the enzyme to the membrane, and the subsequent catalysis. We show that PlsX membrane binding is a process mostly mediated by phospholipid charge, whereas fatty acid saturation and membrane fluidity remarkably influence the membrane insertion step. Strikingly, the PlsXL254E mutant, whose biological functionality was severely compromised in vivo but remains catalytically active in vitro, was able to superficially bind to phospholipid vesicles, nevertheless, it loses the insertion capacity, strongly supporting the importance of membrane insertion in acyl-phosphate delivery. We propose a mechanism in which membrane fluidity governs the insertion of PlsX and thus regulates the biosynthesis of phospholipids in Gram-positive bacteria. This model may be operational in other peripheral membrane proteins with an unprecedented impact in drug discovery/development strategies.