Abstract

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.

Highlights

  • Eukaryotic HMG-CoA reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis [1,2]

  • These studies were done with a crude rabbit polyclonal antibody raised against the catalytic domain of Arabidopsis HMGR1 (Ab-catalytic domain of Arabidopsis HMGR1S (CD1)), but it was later reported that this serum cross-reacts with E. coli proteins [48]

  • Our observations suggest that the HMGR vesicles can measure up to 2 μm in diameter, somewhat more than previously reported

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Summary

Introduction

Eukaryotic HMG-CoA reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis [1,2]. Immunolocalization whole-mount studies in Arabidopsis cotyledon suggested that endogenous HMGR mostly localizes within spherical vesicular structures, which were named HMGR vesicles [18,19]. It is not known how the integral membrane protein HMGR reaches the inside of vesicular structures, nor what relationships exist between these vesicles and the ER. A highly conserved N-terminal motif of plant HMGR is required for OSER biogenesis [19], but no equivalent sequence has been identified in yeast or animal HMGR nor has the morphogenic mechanism been described. We find that the ER-HMGR domains are flexible live entities, fully integrated in ER architecture and dynamism

Subcellular Location of Arabidopsis HMGR in WT and 1S:GFP Transgenic Plants
Reversible Formation of ER-HMGR Domains
The Fixation and Dehydration Method Severely Affects OSER Ultrastructure
Discussion
Plant Material
Constructs
Transient Expression in Nicotiana Benthamiana Leaves
Source and Use of Antibodies
Immunolocalization in Whole Mount
Confocal Microscopy
Chemical Fixation for Ultrastructural Studies
Ultrastructural Analysis
4.10. Immunochemical Ultrastructural Analysis
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