Subcellular Localization of Arabidopsis 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase

Abstract

Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2-to 0.6-mm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR-ER. Nevertheless, they represent a previously undescribed subcellular compartment likely capable of synthesizing mevalonate, which provides new evidence for multiorganelle compartmentalization of the isoprenoid biosynthetic pathways in plants.

Peroxisomal Localization of Arabidopsis Isopentenyl Diphosphate Isomerases Suggests That Part of the Plant Isoprenoid Mevalonic Acid Pathway Is Compartmentalized to Peroxisomes

“…The rate-limiting enzyme, HMGR, has been localized to the ER as well as to unidentified spherical structures (Campos and Boronat, 1995;Leivar et al, 2005), thus mostly conforming to the picture seen in mammals (Goldfarb, 1972). Squalene synthase was shown to be targeted to the ER by a C-terminal hydrophobic trans-membrane domain (Busquets et al, 2008), also conforming to the data from mammalian systems (Stamellos et al, 1993).…”

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

“…HMGR1S transcript is expressed in the whole plant, whereas those encoding HMGR2 or HMGR1L are detected at a much lower level and only in meristems, seedlings, roots, and inflorescences (Enjuto et al, 1994(Enjuto et al, , 1995Lumbreras et al, 1995). Chimeras formed by the membrane domain of plant HMGR fused to GFP localize in the ER network but accumulate in highly fluorescent cytosolic bodies (Leivar et al, 2005;Merret et al, 2007). In this work, we show that these bodies are OSER structures.…”

“…For biolistic transfection, constructs were cloned in plasmid pGFPau (Leivar et al, 2005). For agroinfection or generation of Arabidopsis transgenic plants, constructs were cloned in plasmid pPCV002 (Ferrando et al, 2001) provided by Alejandro Ferrando (Instituto de Biología Molecular y Celular de Plantas, Valencia, Spain).…”

The Remarkable Pliability and Promiscuity of Specialized Metabolism

“…1B). Higher-order metabolons, involving direct physical interactions among enzymes, have also been implicated as key control points in many specialized metabolic pathways, including phenylpropanoid, isoprenoid, alkaloid, and cyanogenic glucoside biosyntheses in plants (Achnine et al 2004;Weid et al 2004;Winkel 2004;Jorgensen et al 2005;Leivar et al 2005;Nielsen et al 2008;Chen et al 2011b). Putative metabolons are postulated to facilitate metabolic channeling, thus exerting a level of kinetic control and specificity by localizing spe- cialized pathways within or on the surface of particular subcellular compartments or by balancing the stoichiometry of participating pathway enzymes.…”