Loose Morphology and High Dynamism of OSER Structures Induced by the Membrane Domain of HMG-CoA Reductase

The membrane domain of eukaryotic HMG-CoA reductase (HMGR) has the conserved capacity to induce endoplasmic reticulum (ER) proliferation and membrane association into Organized Smooth Endoplasmic Reticulum (OSER) structures. These formations develop in response to overexpression of particular proteins, but also occur naturally in cells of the three eukaryotic kingdoms. Here, we characterize OSER structures induced by the membrane domain of Arabidopsis HMGR (1S domain). Immunochemical confocal and electron microscopy studies demonstrate that the 1S:GFP chimera co-localizes with high levels of endogenous HMGR in several ER compartments, such as the ER network, the nuclear envelope, the outer and internal membranes of HMGR vesicles and the OSER structures, which we name ER-HMGR domains. After highpressure freezing, ER-HMGR domains show typical crystalloid, whorled and lamellar ultrastructural patterns, but with wide heterogeneous luminal spaces, indicating that the native OSER is looser and more flexible than previously reported. The formation of ER-HMGR domains is reversible. OSER structures grow by incorporation of ER membranes on their periphery and progressive compaction to the inside. The ER-HMGR domains are highly dynamic in their formation versus their disassembly, their variable spherical-ovoid shape, their fluctuating borders and their rapid intracellular movement, indicating that they are not mere ER membrane aggregates, but active components of the eukaryotic cell

Loose Morphology and High Dynamism of OSER Structures Induced by the Membrane Domain of HMG-CoA Reductase

The membrane domain of eukaryotic HMG-CoA reductase (HMGR) has the conserved capacity to induce endoplasmic reticulum (ER) proliferation and membrane association into Organized Smooth Endoplasmic Reticulum (OSER) structures. These formations develop in response to overexpression of particular proteins, but also occur naturally in cells of the three eukaryotic kingdoms. Here, we characterize OSER structures induced by the membrane domain of Arabidopsis HMGR (1S domain). Immunochemical confocal and electron microscopy studies demonstrate that the 1S:GFP chimera co-localizes with high levels of endogenous HMGR in several ER compartments, such as the ER network, the nuclear envelope, the outer and internal membranes of HMGR vesicles and the OSER structures, which we name ER-HMGR domains. After high-pressure freezing, ER-HMGR domains show typical crystalloid, whorled and lamellar ultrastructural patterns, but with wide heterogeneous luminal spaces, indicating that the native OSER is looser and more flexible than previously reported. The formation of ER-HMGR domains is reversible. OSER structures grow by incorporation of ER membranes on their periphery and progressive compaction to the inside. The ER-HMGR domains are highly dynamic in their formation versus their disassembly, their variable spherical-ovoid shape, their fluctuating borders and their rapid intracellular movement, indicating that they are not mere ER membrane aggregates, but active components of the eukaryotic cell

Identificación, biogénesis y función de Dominios ER-HMGR de retículo endoplasmático en Arabidopsis thaliana

[spa]La sobreexpresión del dominio de membrana de la Hidroximetil glutaril CoA reductasa (HMGR) en Arabidopsis thaliana desencadena la formación de agregados vesiculares de retículo endoplasmático (RE), esta capacidad morfogénica tiene precedentes claros en levaduras y mamíferos. En A. thaliana dos genes (HMG1 y HMG2) codifican tres isoformas de HMGR (HMGR1S, HMGR1L y HMGR2), las proteínas HMGR1S y HMGR1L derivan del mismo gen y tienen la misma secuencia, pero la isoforma 1L tiene una extensión en N-terminal de cincuenta residuos de aminoácidos, las tres isoformas de la HMGR se insertan co-traduccionalmente en la membrana del RE. En la presente tesis se determinó la localización subcelular de la HMGR1S de A. thaliana expresando el dominio de membrana de la HMGR1S unido a GFP, se observó que esta proteína quimérica se localiza en el RE formando agregados, con la ayuda de un marcador rojo T3RE, se observó en ensayos de colocalización en N. benthamiana que los agregados de HMGR son parte funcional del RE. Se determinó la organización estructural de las vesículas de HMGR formadas por el dominio de HMGR1S por primera vez a nivel de ultraestructura en hojas de N. benthamiana, así como también de una línea transgénica de A. thaliana, en este análisis se observaron distintas conformaciones lamelares, de círculos concéntricos y cristaloides presentes en los agregados. Se realizaron ensayos de inmunolocalización a nivel de la ultraestructura de los agregados de 1SGFP en A. thaliana, con anticuerpos contra GFP y anticuerpos contra el dominio catalítico de la HMGR, y se observó el marcaje de ambos anticuerpos, indicándonos la presencia en estos agregados, tanto de la 1SGFP como de la HMGR endógena, confirmando que el dominio de membrana de HMGR determina su localización subcelular. Los dominios de RE en el que se acumulan 1S:GFP y/o HMGR fueron denominados como Dominios ER-HMGR. Por otro lado, a pesar de las dificultades técnicas, un estudio anterior sugiere la coincidencia física entre vesículas de HMGR y ER bodies, estas últimas son estructuras derivadas de retículo endoplasmático que cumplen un rol importante en el mecanismo de defensa de las plantas en condiciones de estrés. La identidad estructural y funcional de los ER bodies no ha sido caracterizada aún. En este sentido en la presente tesis se profundizo en el estudio de la coincidencia física y funcional entre vesículas de HMGR y ER bodies, como su caracterización estructural en distintos tipos celulares de A. thaliana. Además, se determinó el papel biológico del dominio de membrana de HMGR en respuesta a estrés utilizando líneas mutantes knock out para el gen HMG1. En este análisis se observó que el gen de la HMGR1 es necesario para la derivación de Dominos ER-HMGR hacia vacuola en respuesta a estrés.[eng]The overexpression of hydroxymetyl-glutaryl CoA reductase (HMGR) membrane domain in Arabidopsis thaliana triggers the endoplasmic reticulum (ER) vesicular aggregates formation, this morphogenetic capability has clear precedents in yeast and mammal cells. In the present thesis, the subcellular localization of A. thaliana HMGR1S isoform was determined through the expression of the HMGR1S membrane domain attached to GFP. This chimerical protein 1S:GFP is localized in the ER forming aggregates, the use of a red lumen ER marker T3RE allowed through colocalization assays in N. benthamiana, determine the 1S:GFP aggregates are a functional part of the ER. The ultra-structural organization of 1S:GFP aggregates induced by the HMGR membrane domain expression was determined for the first time at this level, in N. benthamiana agroinfiltrated and transgenic lines of A. thaliana. In this analysis, was observed that the 1S:GFP aggregates are conformed by smooth ER membranes distributed with different organization patterns, such as lamellar, whorls and mainly crystalloid. The immuno localization assays of 1S:GFP aggregates in A. thaliana at ultra-structural level, with anti-GFP and anti-CD1 (CD1:catalytic domain of HMGR1), revealed the presence in ER aggregates of 1S:GFP and HMGR distributed homogenously, therefore these aggregates are a specific domain characterized by the presence of HMGR and denominated as ER-HMGR domain. On the other hand, a previous study suggested the physical coincidence between HMGR vesicles and ER bodies, the last ones are ER derived structures which accumulates specific hydrolytic enzymes and has an important role in plant defense against stress conditions and pathogen attack. In the present work, the study of the physical coincidence between HMGR vesicles and ER bodies and the structural characterization in different tissues and cells was detailed. Additionally, the biologic role of the HMGR1S membrane domain in stress conditions was determined, using HMG1 gene knock out lines. In this analysis it was observed that the HMG1 gene is necessary to derive ER-HMGR domains to vacuole in response to stress conditions

Proliferation and Morphogenesis of the Endoplasmic Reticulum Driven by the Membrane Domain of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in Plant Cells

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis and is composed of an endoplasmic reticulum (ER)-anchoring membrane domain with low sequence similarity among eukaryotic kingdoms and a conserved cytosolic catalytic domain. Organized smooth endoplasmic reticulum (OSER) structures are common formations of hypertrophied tightly packed ER membranes devoted to specific biosynthetic and secretory functions, the biogenesis of which remains largely unexplored. We show that the membrane domain of plant HMGR suffices to trigger ER proliferation and OSER biogenesis. The proliferating membranes become highly enriched in HMGR protein, but they do not accumulate sterols, indicating a morphogenetic rather than a metabolic role for HMGR. The N-terminal MDVRRRPP motif present in most plant HMGR isoforms is not required for retention in the ER, which was previously proposed, but functions as an ER morphogenic signal. Plant OSER structures are morphologically similar to those of animal cells, emerge from tripartite ER junctions, and mainly build up beside the nuclear envelope, indicating conserved OSER biogenesis in high eukaryotes. Factors other than the OSER-inducing HMGR construct mediate the tight apposition of the proliferating membranes, implying separate ER proliferation and membrane association steps. Overexpression of the membrane domain of Arabidopsis (Arabidopsis thaliana) HMGR leads to ER hypertrophy in every tested cell type and plant species, whereas the knockout of the HMG1 gene from Arabidopsis, encoding its major HMGR isoform, causes ER aggregation at the nuclear envelope. Our results show that the membrane domain of HMGR contributes to ER morphogenesis in plant cells