Endoplasmic Reticulum Export of GPI-Anchored Proteins

Protein export from the endoplasmic reticulum (ER) is an essential process in all eukaryotes driven by the cytosolic coat complex COPII, which forms vesicles at ER exit sites for transport of correctly assembled secretory cargo to the Golgi apparatus. The COPII machinery must adapt to the existing wide variety of different types of cargo proteins and to different cellular needs for cargo secretion. The study of the ER export of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), a special glycolipid-linked class of cell surface proteins, is contributing to address these key issues. Due to their special biophysical properties, GPI-APs use a specialized COPII machinery to be exported from the ER and their processing and maturation has been recently shown to actively regulate COPII function. In this review, we discuss the regulatory mechanisms by which GPI-APs are assembled and selectively exported from the ER.Ministerio de Economía y Competitividad de España (MINECO)-BFU2017-89700-

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. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins.FEDER/Ministerio de Ciencia, Innovación y Universidades BFU2017-89700-PJapan Society for the Promotion of Science JP19H0292

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. To overcome this technical challenge, we have applied a crosslinking assay to stabilize the transient and/or weak protein interactions. Here, we describe a protocol of protein crosslinking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system

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. Here, we show biochemically that the p24 complex associates at the ER with other cargo receptors in a COPII-dependent manner, forming high-molecular weight multireceptor complexes. Furthermore, live cell imaging analysis reveals that the p24 complex is required to retain in the ER secretory cargos when their specific receptors are absent. This requirement does not involve neither the unfolded protein response nor the retrograde transport from the Golgi. Our results suggest that, in addition to its role as a cargo receptor in the specialized GPI-anchored protein pathway, the p24 complex also plays an independent role in secretory cargo selectivity during its exit through the general ER export pathway, preventing the non-selective bulk flow of native secretory cargos. This mechanism would ensure receptor-regulated cargo transport, providing an additional layer of regulation of secretory cargo selectivity during ER export.Ministerio de Economía y Competitividad BFU2017-89700-P, BFU2016-78265-

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. Furthermore, we show that the chain length of ceramide in the endoplasmic reticulum membrane is critical for this sorting selectivity. Our study provides the first direct in vivo evidence for lipid chain length–based protein cargo sorting into selective export sites of the secretory pathway.Spanish Ministry of Economy and Competitiveness (MINECO; grant number BFU2017-89700-P)University of Seville (VIPPIT-2020-I.5)Japan Society for the Promotion of Science (JP25221103, JP17H06420, and JP18H05275)Swiss National Science Foundation (grant no. 163966)Swiss National Supercomputing Centre (CSCS) under project IDs s726 and s84

Ahora / Ara

La cinquena edició del microrelatari per l’eradicació de la violència contra les dones de l’Institut Universitari d’Estudis Feministes i de Gènere «Purificación Escribano» de la Universitat Jaume I vol ser una declaració d’esperança. Aquest és el moment en el qual les dones (i els homes) hem de fer un pas endavant i eliminar la violència sistèmica contra les dones. Ara és el moment de denunciar el masclisme i els micromasclismes començant a construir una societat més igualitària. Cadascun dels relats del llibre és una denúncia i una declaració que ens encamina cap a un món millor

Role of Lipids in Protein Sorting From the Endoplasmic Reticulum

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 long-standing fundamental question that still remains unanswered. The present thesis aimed to directly address this central issue by investigating in yeast whether and how specific membrane lipids are involved in differential protein export from the endoplasmic reticulum (ER), the first essential transport step of the secretory pathway. We conduct 3D simultaneous multi-color high resolution live imaging to demonstrate that newly synthesized lipid-linked plasma membrane proteins, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) having a very long chain ceramide (C26) lipid moiety, are clustered and sorted into specialized endoplasmic reticulum exit sites (ERES) that are distinct from those used by transmembrane plasma membrane proteins during their export from the ER. Furthermore, we show that the chain length (C26) of ceramide in the ER membrane is critical for this sorting selectivity. We also show that, in addition to the presence of C26 ceramide in the ER membrane, clustering of GPI-APs requires binding to the transmembrane p24 protein complex, the cargo receptor that specifically exports GPI-APs from the ER. Using molecular dynamics simulations, we found that membrane ceramides accumulate around the cytosolic leaflet of the p24 transmembrane helix. Based on our results we propose a mechanism for C26 ceramide-based sorting by which the local enrichment of C26 ceramide in both leaflets of the ER membrane upon multivalent p24 binding of GPI-APs with C26 ceramide in the luminal leaflet and p24 association of free C26 membrane ceramide in the cytosolic leaflet could promote concomitant protein clustering and membrane curvature by C26 acyl chain interdigitation. In sum, our study provides in vivo evidence for lipid chain length-based protein cargo sorting into selective export sites of the secretory pathway and better comprehension of potential mechanism for this lipid-based sorting

Diseño de un compactador de PET de uso ligero

Tesina (Ingeniería en Comunicaciones y Electrónica), Instituto Politécnico Nacional, ESIME, Unidad Culhuacán, 2008, 1 archivo PDF, (141 páginas). tesis.ipn.m

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). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37°C). ER export is restored by shifting down to permissive temperature (24°C) and progressive incorporation of the two different types of cargos into the fluorescently labelled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide present in the ER membrane is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES

Violencia escolar

Tesis (Profesor de Educación Física para la Enseñanza Básica)El trabajo que presentamos a continuación, tratará de describir las manifestaciones de violencia y agresividad que presentan los alumnos de los colegios corporizados de la comuna de Viña del Mar, tomando en cuenta los niveres básicos uno y dos. Nos referimos como manifestaciones de agresividad y violencia infantil a aquellas que se presentan en la convivencia diaria con pares, profesores y hermanos