Impact of sphingolipids on protein membrane trafficking.

Membrane trafficking is essential to maintain the spatiotemporal control of protein and lipid distribution within membrane systems of eukaryotic cells. To achieve their functional destination proteins are sorted and transported into lipid carriers that construct the secretory and endocytic pathways. It is an emerging theme that lipid diversity might exist in part to ensure the homeostasis of these pathways.

GPI anchors: Regulated as needed.

GPI anchoring is an essential post-translational modification in eukaryotes that links proteins to the plasma membrane. In this issue, Liu et al. (2023.

Quality-controlled ceramide-based GPI-anchored protein sorting into selective ER exit sites.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) exit the endoplasmic reticulum (ER) through a specialized export pathway in the yeast Saccharomyces cerevisiae. We have recently shown that a very-long acyl chain (C26) ceramide present in the ER membrane drives clustering and sorting of GPI-APs into selective ER exit sites (ERES). Now, we show that this lipid-based ER sorting also involves the C26 ceramide as a lipid moiety of GPI-APs, which is incorporated into the GPI anchor through a lipid-remodeling process after protein attachment in the ER.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway.

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation.

The Ceramide Synthase Subunit Lac1 Regulates Cell Growth and Size in Fission Yeast.

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast.

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.

Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).

Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM).

Structural analysis of the GPI glycan.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion.

Determination of the lipid composition of the GPI anchor.

In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release and extraction of the GPI-lipid moiety and analysis by mass spectrometry.

The p24 Complex Contributes to Specify Arf1 for COPI Coat Selection.

Golgi trafficking depends on the small GTPase Arf1 which, upon activation, drives the assembly of different coats onto budding vesicles. Two related types of guanine nucleotide exchange factors (GEFs) activate Arf1 at different Golgi sites. In yeast, Gea1 in the -Golgi and Gea2 in the medial-Golgi activate Arf1 to form COPI-coated vesicles for retrograde cargo sorting, whereas Sec7 generates clathrin/adaptor-coated vesicles at the -Golgi network (TGN) for forward cargo transport.

Ceramide chain length-dependent protein sorting into selective endoplasmic reticulum exit sites.

Protein sorting in the secretory pathway is crucial to maintain cellular compartmentalization and homeostasis. In addition to coat-mediated sorting, the role of lipids in driving protein sorting during secretory transport is a longstanding fundamental question that still remains unanswered. Here, we conduct 3D simultaneous multicolor high-resolution live imaging to demonstrate in vivo that newly synthesized glycosylphosphatidylinositol-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized endoplasmic reticulum exit sites that are distinct from those used by transmembrane proteins.

Dual Independent Roles of the p24 Complex in Selectivity of Secretory Cargo Export from the Endoplasmic Reticulum.

The cellular mechanisms that ensure the selectivity and fidelity of secretory cargo protein transport from the endoplasmic reticulum (ER) to the Golgi are still not well understood. The p24 protein complex acts as a specific cargo receptor for GPI-anchored proteins by facilitating their ER exit through a specialized export pathway in yeast. In parallel, the p24 complex can also exit the ER using the general pathway that exports the rest of secretory proteins with their respective cargo receptors.

Yeast ceramide synthases, Lag1 and Lac1, have distinct substrate specificity.

was the first longevity assurance gene discovered in The Lag1 protein is a ceramide synthase and its homolog, Lac1, has a similar enzymatic function but no role in aging. Lag1 and Lac1 lie in an enzymatic branch point of the sphingolipid pathway that is interconnected by the activity of the C4 hydroxylase, Sur2. By uncoupling the enzymatic branch point and using lipidomic mass spectrometry, metabolic labeling and assays we show that Lag1 preferentially synthesizes phyto-sphingolipids.

Limited ER quality control for GPI-anchored proteins.

Endoplasmic reticulum (ER) quality control mechanisms target terminally misfolded proteins for ER-associated degradation (ERAD). Misfolded glycophosphatidylinositol-anchored proteins (GPI-APs) are, however, generally poor ERAD substrates and are targeted mainly to the vacuole/lysosome for degradation, leading to predictions that a GPI anchor sterically obstructs ERAD. Here we analyzed the degradation of the misfolded GPI-AP Gas1* in yeast.

Intracellular sphingosine releases calcium from lysosomes.

To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release.

COPII coat composition is actively regulated by luminal cargo maturation.

Background: Export from the ER is an essential process driven by the COPII coat, which forms vesicles at ER exit sites (ERESs) to transport mature secretory proteins to the Golgi. Although the basic mechanism of COPII assembly is known, how COPII machinery is regulated to meet varying cellular secretory demands is unclear.

Results: Here, we report a specialized COPII system that is actively recruited by luminal cargo maturation.

Osh proteins regulate COPII-mediated vesicular transport of ceramide from the endoplasmic reticulum in budding yeast.

Lipids synthesized at the endoplasmic reticulum (ER) are delivered to the Golgi by vesicular and non-vesicular pathways. ER-to-Golgi transport is crucial for maintaining the different membrane lipid composition and identities of organelles. Despite their importance, mechanisms regulating transport remain elusive.

Sphingolipid homeostasis in the web of metabolic routes.

Sphingolipids play a key role in cells as structural components of membrane lipid bilayers and signaling molecules implicated in important physiological and pathological processes. Their metabolism is tightly regulated. Mechanisms controlling sphingolipid metabolism are far from being completely understood.

The yeast p24 complex regulates GPI-anchored protein transport and quality control by monitoring anchor remodeling.

Glycosylphosphatidylinositol (GPI)-anchored proteins are secretory proteins that are attached to the cell surface of eukaryotic cells by a glycolipid moiety. Once GPI anchoring has occurred in the lumen of the endoplasmic reticulum (ER), the structure of the lipid part on the GPI anchor undergoes a remodeling process prior to ER exit. In this study, we provide evidence suggesting that the yeast p24 complex, through binding specifically to GPI-anchored proteins in an anchor-dependent manner, plays a dual role in their selective trafficking.

The yeast p24 complex is required for the formation of COPI retrograde transport vesicles from the Golgi apparatus.

The p24 family members are transmembrane proteins assembled into heteromeric complexes that continuously cycle between the ER and the Golgi apparatus. These cargo proteins were assumed to play a structural role in COPI budding because of their major presence in mammalian COPI vesicles. However, this putative function has not been proved conclusively so far.