The p97-UBXD8 complex regulates ER-Mitochondria contact sites by altering membrane lipid saturation and composition.

The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-Mitochondria contact sites (ERMCS) is a platform for critical cellular processes, particularly lipid synthesis. How contacts are remodeled and the impact of altered contacts on lipid metabolism remains poorly understood. We show that the p97 AAA-ATPase and its adaptor ubiquitin-X domain adaptor 8 (UBXD8) regulate ERMCS. The p97-UBXD8 complex localizes to contacts and its loss increases contacts in a manner that is dependent on p97 catalytic activity. Quantitative proteomics and lipidomics of ERMCS demonstrates alterations in proteins regulating lipid metabolism and a significant change in membrane lipid saturation upon UBXD8 deletion. Loss of p97-UBXD8 increased membrane lipid saturation via SREBP1 and the lipid desaturase SCD1. Aberrant contacts can be rescued by unsaturated fatty acids or overexpression of SCD1. We find that the SREBP1-SCD1 pathway is negatively impacted in the brains of mice with p97 mutations that cause neurodegeneration. We propose that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation.

Human Cytomegalovirus Uses a Host Stress Response To Balance the Elongation of Saturated/Monounsaturated and Polyunsaturated Very-Long-Chain Fatty Acids.

Stress and virus infection regulate lipid metabolism. Human cytomegalovirus (HCMV) infection induces fatty acid (FA) elongation and increases the abundance of lipids with very-long-chain FA (VLCFA) tails. While reprogramming of metabolism can be stress related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knock out protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. In HCMV-infected cells, PERK promotes increases in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFA tails. Further, PERK enhances FA elongase 7 (ELOVL7) protein levels, which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection were independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Additionally, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK-and, more broadly, ER stress-may be necessary for the membrane biogenesis needed to generate infectious HCMV virions.IMPORTANCE HCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator PERK controls FA elongation and the cellular abundance of several types of lipids following HCMV infection. Specifically, PERK promotes FA elongase 7 synthesis and phospholipids with saturated/monounsaturated very-long-chain FA tails. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.

Methodological Approaches Frame Insights into Endophyte Richness and Community Composition.

Isolating microbes is vital to study microbiomes, but insights into microbial diversity and ecology can be constrained by recalcitrant or unculturable strains. Culture-free methods (e.g., next-generation sequencing, NGS) have become popular in part because they detect greater richness than culturing alone. Both approaches are used widely to characterize microfungi within healthy leaves (foliar endophytes), but methodological differences among studies can constrain large-scale insights into endophyte ecology. We examined endophytes in a temperate plant community to quantify how certain methodological factors, such as the choice of cultivation media for culturing and storage period after leaf collection, affect inferences regarding endophyte communities; how such effects vary among plant taxa; and how complementary culturing and NGS can be when subsets of the same plant tissue are used for each. We found that endophyte richness and composition from culturing were consistent across five media types. Insights from culturing and NGS were largely robust to differences in storage period (1, 5, and 10 days). Although endophyte richness, composition, and taxonomic diversity identified via culturing vs. NGS differed markedly, both methods revealed host-structured communities. Studies differing only in cultivation media or storage period thus can be compared to estimate endophyte richness, composition, and turnover at scales larger than those of individual studies alone. Our data show that it is likely more important to sample more host species, rather than sampling fewer species more intensively, to quantify endophyte diversity in given locations, with the richest insights into endophyte ecology emerging when culturing and NGS are paired.