Manuel Muñiz

Protein sorting upon exit from the endoplasmic reticulum.

It is currently thought that all secretory proteins travel together to the Golgi apparatus where they are sorted to different destinations. However, the specific requirements for transport of GPI-anchored proteins from the endoplasmic reticulum to the Golgi apparatus in yeast could be explained if protein sorting occurs earlier in the pathway. Using an in vitro assay that reconstitutes a single round of budding from the endoplasmic reticulum, we found that GPI-anchored proteins and other secretory proteins exit the endoplasmic reticulum in distinct vesicles. Therefore, GPI-anchored proteins are sorted from other proteins, in particular other plasma membrane proteins, at an early stage of the secretory pathway. These results have wide implications for the mechanism of protein exit from the endoplasmic reticulum.

Intracellular transport of GPI‐anchored proteins

In eukaryotic cells, a subset of proteins are attached to the external leaflet of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. There is substantial evidence suggesting that these GPI‐anchored proteins are clustered in sphingolipid–sterol microdomains or rafts. Since the precursors of these microdomain components are synthesized mainly in the endoplasmic reticulum, it is possible that microdomain assembly occurs during transport along the exocytic route. A sorting mechanism for GPI‐anchored proteins using sphingolipid microdomains as selective platforms for vesicle budding has been proposed to operate at different steps in the secretory pathway. Here, we discuss this sorting model in the context of the data obtained from different biological and artificial systems, in addition to other particularities of the intracellular transport of the GPI‐anchored proteins.

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

Two functions of the p24 complex are described: one connects GPI-anchored proteins to COPII proteins at ER exit sites to facilitate their incorporation into ER-derived vesicles, and the other serves in quality control of GPI-anchored proteins to retrieve unremodeled GPI-anchored proteins from the Golgi back to the ER.

A Regulatory Role for cAMP-dependent Protein Kinase in Protein Traffic along the Exocytic Route

The influence of protein kinase A activity on transport of newly synthesized vesicular stomatitis virus G glycoprotein along the exocytic pathway was examined. Transport of vesicular stomatitis virus G glycoprotein to the cell surface was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a selective inhibitor of protein kinase A. This block occurred at the exit of the Golgi complex, whereas transport through the Golgi compartments or from the endoplasmic reticulum to the Golgi was decreased in the presence of H-89. As judged by immunofluorescence endoplasmic reticulum to Golgi transport was accelerated in cells incubated with activators of protein kinase A such as isobutylmethylxanthine (IBMX) or forskolin (FK). Treatment with IBMX and FK also increased transport from the trans-Golgi network to the cell surface. During incubation with IBMX and FK, the organization of the Golgi complex was altered showing intercisternae fusion and miscompartmentalization of resident proteins. These structural changes affected both the kinetics of acquisition of endoglycosidase H resistance and transport activities. These data support a differential regulatory role for protein kinase A in different transport steps along the exocytic pathway. In particular, transport from the trans-Golgi network to the cell surface was dependent on protein kinase A activity. In addition, the results suggest the involvement of this enzyme on the maintenance of the Golgi complex organization.

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. Furthermore, deletion of all eight yeast p24 family members does not produce severe transport phenotypes, suggesting that the p24 complex is not essential for COPI function. In this paper we provide direct evidence that the yeast p24 complex plays an active role in retrograde transport from Golgi to ER by facilitating the formation of COPI-coated vesicles. Therefore, our results demonstrate that p24 proteins are important for vesicle formation instead of simply being a passive traveler, supporting the model in which cargo together with a small GTPase of the ARF superfamily and coat subunits act as primer for vesicle formation.

Sorting of GPI-anchored proteins from yeast to mammals – common pathways at different sites?

ABSTRACT Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are luminal secretory cargos that are attached by a post-translational glycolipid modification, the GPI anchor, to the external leaflet of the plasma membrane. GPI-APs are conserved among eukaryotes and possess many diverse and vital functions for which the GPI membrane attachment appears to be essential. The presence of the GPI anchor and its subsequent modifications along the secretory pathway confer to the anchored proteins unique trafficking properties that make GPI-APs an exceptional system to study mechanisms of sorting. In this Commentary, we discuss the recent advances in the field of GPI-AP sorting focusing on the mechanisms operating at the level of the exit from the ER and from the trans-Golgi network (TGN), which take place, respectively, in yeast and in polarized mammalian cells. By considering the similarities and differences between these two sorting events, we present unifying principles that appear to work at different sorting stations and in different organisms.

Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells.

Sphingolipids are not only important components of membranes but also have functions in protein trafficking and intracellular signaling. The LCB1 gene encodes a subunit of the serine palmitoyltransferase, which is responsible for the first step of sphingolipid synthesis. Here, we show that activation of the unfolded protein response (UPR) can restore normal ceramide levels and viability in yeast cells with a conditional defect in LCB1. Dependence on UPR was demonstrated by showing the HAC1-dependence of the suppression. A similar induction of ceramides by UPR seems to take place in mammalian cells. In rat pancreatic INS-1E cells, UPR activation induces the transcription of the CerS6 gene, which encodes a ceramide synthase. This correlates with the specific accumulation of ceramide with a C16 fatty acyl chain upon UPR activation. Therefore, our study reveals a novel connection between UPR induction and ceramide synthesis that seems to be conserved between yeast and mammalian cells.

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. Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are luminal secretory proteins anchored to the membrane by the glycolipid GPI. After protein attachment in the ER lumen, lipid and glycan parts of the GPI anchor are remodeled. In yeast, GPI-lipid remodeling concentrates GPI-APs into specific ERESs. We found that GPI-glycan remodeling induces subsequent recruitment of the specialized ER export machinery that enables vesicle formation from these specific ERESs. First, the transmembrane cargo receptor p24 complex binds GPI-APs as a lectin by recognizing the remodeled GPI-glycan. Binding of remodeled cargo induces the p24 complex to recruit the COPII subtype Lst1p, specifically required for GPI-AP ER export. CONCLUSIONS Our results show that COPII coat recruitment by cargo receptors is not constitutive but instead is actively regulated by binding of mature ligands. Therefore, we reveal a novel functional link between luminal cargo maturation and COPII vesicle budding, providing a mechanism to adjust specialized COPII vesicle production to the amount and quality of their luminal cargos that are ready for ER exit. This helps to understand how the ER export machinery adapts to different needs for luminal cargo secretion.

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

ABSTRACT 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. Here we report that in yeast coat protein complex II (COPII) vesicle-mediated transport of ceramide from the ER to the Golgi requires oxysterol-binding protein homologs, Osh proteins, which have been implicated in lipid homeostasis. Because Osh proteins are not required to transport proteins to the Golgi, these results indicate a specific requirement for the Osh proteins in the transport of ceramide. In addition, we provide evidence that Osh proteins play a negative role in COPII vesicle biogenesis. Together, our data suggest that ceramide transport and sphingolipid levels between the ER and Golgi are maintained by two distinct functions of Osh proteins, which negatively regulate COPII vesicle formation and positively control a later stage, presumably fusion of ceramide-enriched vesicles with Golgi compartments.

Trafficking of glycosylphosphatidylinositol anchored proteins from the endoplasmic reticulum to the cell surface

In eukaryotes, many cell surface proteins are attached to the plasma membrane via a glycolipid glycosylphosphatidylinositol (GPI) anchor. GPI-anchored proteins (GPI-APs) receive the GPI anchor as a conserved posttranslational modification in the lumen of the endoplasmic reticulum (ER). After anchor attachment, the GPI anchor is structurally remodeled to function as a transport signal that actively triggers the delivery of GPI-APs from the ER to the plasma membrane, via the Golgi apparatus. The structure and composition of the GPI anchor confer a special mode of interaction with membranes of GPI-APs within the lumen of secretory organelles that lead them to be differentially trafficked from other secretory membrane proteins. In this review, we examine the mechanisms by which GPI-APs are selectively transported through the secretory pathway, with special focus on the recent progress made in their actively regulated export from the ER and the trans-Golgi network.