A Role for Lipids in Protein Sorting?

Lipid and protein diversity provides structural and functional identity to the membrane compartments that define the eukaryotic cell. This compositional heterogeneity is maintained by the secretory pathway, which feeds newly synthesized proteins and lipids to the endomembrane systems. The precise sorting of lipids and proteins through the pathway guarantees the achievement of their correct delivery. Although proteins have been shown to be key for sorting mechanisms, whether and how lipids contribute to this process is still an open discussion. Our laboratory, in collaboration with other groups, has recently addressed the long-postulated role of membrane lipids in protein sorting in the secretory pathway, by investigating in yeast how a special class of lipid-linked cell surface proteins are differentially exported from the endoplasmic reticulum. Here we comment on this interdisciplinary study that highlights the role of lipid diversity and the importance of protein-lipid interactions in sorting processes at the cell membrane.

Structural organization of erythrocyte membrane microdomains and their relation with malaria susceptibility

Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P. falciparum invasion. We also found that genetic variants in both ART4 and AQP1 are associated with susceptibility to the disease in a malaria-endemic population.

Crossroads between membrane trafficking machinery and copper homeostasis in the nerve system

Imbalanced copper homeostasis and perturbation of membrane trafficking are two common symptoms that have been associated with the pathogenesis of neurodegenerative and neurodevelopmental diseases. Accumulating evidence from biophysical, cellular and in vivo studies suggest that membrane trafficking orchestrates both copper homeostasis and neural functions—however, a systematic review of how copper homeostasis and membrane trafficking interplays in neurons remains lacking. Here, we summarize current knowledge of the general trafficking itineraries for copper transporters and highlight several critical membrane trafficking regulators in maintaining copper homeostasis. We discuss how membrane trafficking regulators may alter copper transporter distribution in different membrane compartments to regulate intracellular copper homeostasis. Using Parkinson's disease and MEDNIK as examples, we further elaborate how misregulated trafficking regulators may interplay parallelly or synergistically with copper dyshomeostasis in devastating pathogenesis in neurodegenerative diseases. Finally, we explore multiple unsolved questions and highlight the existing challenges to understand how copper homeostasis is modulated through membrane trafficking.

Influence of the Position of Mango Fruit on the Tree (Mangifera indica L. CV. ‘Zibda’) on Chilling Sensitivity and Antioxidant Enzyme Activity

Mango fruits sourced from tropical yields have had a high commercial comeback from being viewed as susceptible to chilling injury under long storage durations. When the fruits are exposed to cold storage, this results in physiological changes due to the side effects of the storage on the fruits, expanding the rates of loss during the period between harvest and marketing. It is difficult to harvest mangoes as the fruits show varying maturities and are located in different positions on the trees. The purpose of this study was to test the idea that fruits’ location on the tree influences how the fruit behaves during cold storage. During two seasons (2019–2020), the impact of on-tree fruit location, i.e., sunny side (SUN; fruit exposed to the sun for most of the day), shade (SHA; fruit grown on the shady side of trees), and inside the canopy (INS; fruit grown inside the tree canopy), on the chilling sensitivity and the activities of antioxidant enzymes of ‘Zibda’ mangos stored at a low temperature (4 ± 1 °C) for 35 days was determined. In contrast to SHA and SUN mangos, INS fruits were shown to be progressively tolerant to low storage temperatures. These fruits also showed the highest activities of ascorbate peroxidase (APX), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD). In addition, the contents of O2− and H2O2 decreased in INS fruit during storage. Consequently, the cell membrane compartments were maintained, showing low accumulation of both malondialdehyde (MDA) and the protein carbonyl group (PCG) during storage. These results indicate that the fruit positions can also be considered at the time of harvesting for the classification of fruits before cold storage. This classification can also be added to the mango trading protocol to minimize the loss of economic returns by chilling injury.

Data independent acquisition enables deep and fast label-free dynamic organellar mapping

The membrane compartments of eukaryotic cells organize the proteome into dynamic reaction spaces that control protein activity. This 'spatial proteome' and its changes can be captured systematically by our previously established Dynamic Organellar Maps (DOMs) approach, which combines cell fractionation and shotgun-proteomics into a profiling analysis of subcellular localization. Our original method relied on data dependent acquisition (DDA), which is inherently stochastic, and thus offers limited depth of analysis across replicates. Here we adapt DOMs to data independent acquisition (DIA), in a label-free format, and establish an automated data quality control tool to benchmark performance. Matched for mass spectrometry (MS) runtime, DIA-DOMs provide double the depth relative to DDA-DOMs, with substantially improved precision and localization prediction performance. Matched for depth, DIA-DOMs provide organellar maps in a third of the runtime. To test the DIA-DOMs performance for comparative applications, we mapped subcellular localization changes in response to starvation/disruption of lysosomal pH in HeLa cells, revealing a subset of Golgi proteins that cycle through endosomes. DIA-DOMs offer a superior workflow for label-free spatial proteomics, with a broad application spectrum in cell and biomedical research.

PDZD-8 and TEX-2 regulate endosomal PI(4,5)P2 homeostasis via lipid transport to promote embryogenesis in C. elegans

Different types of cellular membranes have unique lipid compositions that are important for their functional identity. PI(4,5)P2 is enriched in the plasma membrane where it contributes to local activation of key cellular events, including actomyosin contraction and cytokinesis. However, how cells prevent PI(4,5)P2 from accumulating in intracellular membrane compartments, despite constant intermixing and exchange of lipid membranes, is poorly understood. Using the C. elegans early embryo as our model system, we show that the evolutionarily conserved lipid transfer proteins, PDZD-8 and TEX-2, act together with the PI(4,5)P2 phosphatases, OCRL-1 and UNC-26/synaptojanin, to prevent the build-up of PI(4,5)P2 on endosomal membranes. In the absence of these four proteins, large amounts of PI(4,5)P2 accumulate on endosomes, leading to embryonic lethality due to ectopic recruitment of proteins involved in actomyosin contractility. PDZD-8 localizes to the endoplasmic reticulum and regulates endosomal PI(4,5)P2 levels via its lipid harboring SMP domain. Accumulation of PI(4,5)P2 on endosomes is accompanied by impairment of their degradative capacity. Thus, cells use multiple redundant systems to maintain endosomal PI(4,5)P2 homeostasis.

Tril dampens Nodal signaling through Pellino2- and Traf6-mediated activation of Nedd4l

Toll-like receptor 4 (Tlr) interactor with leucine-rich repeats (Tril) functions as a Tlr coreceptor to mediate innate immunity in adults. In Xenopus embryos, Tril triggers degradation of the transforming growth factor β (Tgf-ß) family inhibitor, Smad7. This enhances bone morphogenetic protein (Bmp) signaling to enable ventral mesoderm to commit to a blood fate. Here, we show that Tril simultaneously dampens Nodal signaling by catalytically activating the ubiquitin ligase NEDD4 Like (Nedd4l). Nedd4l then targets Nodal receptors for degradation. How Tril signals are transduced in a nonimmune context is unknown. We identify the ubiquitin ligase Pellino2 as a protein that binds to the cytoplasmic tail of Tril and subsequently forms a complex with Nedd4l and another E3 ligase, TNF-receptor associated factor 6 (Traf6). Pellino2 and Traf6 are essential for catalytic activation of Nedd4l, both in Xenopus and in mammalian cells. Traf6 ubiquitinates Nedd4l, which is then recruited to membrane compartments where activation occurs. Collectively, our findings reveal that Tril initiates a noncanonical Tlr-like signaling cascade to activate Nedd4l, thereby coordinately regulating the Bmp and Nodal arms of the Tgf-ß superfamily during vertebrate development.

Nano-scale resolution of native retinal rod disk membranes reveals differences in lipid composition

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample's copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein–lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.