**1. Introduction**

Eukaryotic HMG-CoA reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis [1,2]. Isoprenoid products derived from this pathway are required for many diverse essential functions, including membrane biogenesis (sterols), control of growth and development (steroid hormones and plant cytokinins), protein prenylation (farnesyl and geranyl groups), protein glycosylation (dolichols) and respiration (ubiquinones) [3]. In plants, the mevalonate pathway also provides a wide variety of secondary metabolites required for defence against herbivores and pathogens or for the attraction of beneficial organisms [4]. In all plant species, HMGR is encoded by a multigene family. This was first proposed after analysis of few model plants [5], but has been confirmed by high throughput sequencing of an ever-increasing number of genomes [6]. In *Arabidopsis thaliana*, two genes (*HMG1* and *HMG2*) encode three HMGR isoforms (HMGR1S, HMGR1L and HMGR2) [5,7]. It has been suggested that different variants of plant HMGR

**Citation:** Grados-Torrez, R.E.; López-Iglesias, C.; Ferrer, J.C.; Campos, N. Loose Morphology and High Dynamism of OSER Structures Induced by the Membrane Domain of HMG-CoA Reductase. *Int. J. Mol. Sci.* **2021**, *22*, 9132.

https://doi.org/10.3390/ ijms22179132

Academic Editor: Masoud Jelokhani-Niaraki

Received: 18 July 2021 Accepted: 21 August 2021 Published: 24 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

are physically associated with other enzymes, forming metabolons for the synthesis of particular isoprenoid products, and that these metabolons would be located at particular sites of the endomembrane system [8]. The association of sterol biosynthetic enzymes at the ER membrane (one of the branches of the isoprenoid pathway) has been shown in yeast, mammals and plants [9–11], but no proof of the existence of metabolons involving HMGR has ye<sup>t</sup> been provided.

HMGR is composed of an N-terminal membrane domain, with low or no sequence similarity among eukaryotic kingdoms, and a highly conserved catalytic domain [12–14]. In plant HMGR, the membrane domain has only two hydrophobic segments, whereas in yeas<sup>t</sup> and animal HMGR eight membrane-spanning regions have been predicted [12,15,16]. The three *Arabidopsis* HMGR isoforms are primarily targeted to the ER by the two hydrophobic sequences of the membrane domain that interact specifically with the Signal Recognition Particle [12,17]. However, immunolocalization whole-mount studies in *Arabidopsis* cotyledon suggested that endogenous HMGR mostly localizes within spherical vesicular structures, which were therefore 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.

Despite diverging evolution, the membrane domain of HMGR from the three eukaryotic kingdoms has the common capacity to induce massive proliferation of ER membranes that subsequently constitute Organized Smooth Endoplasmic Reticulum (OSER) structures [19–21]. When examined by transmission electron microscopy (EM), OSER structures contain tightly associated ER membranes according to three different patterns: ordered arrays with hexagonal or cubic symmetry (crystalloid ER), concentric layers (whorled ER) or simply stacked (lamellae or perinuclear karmellae) [21–23]. A highly conserved N-terminal motif of plant HMGR is required for OSER biogenesis [19], but no equivalent sequence has been identified in yeas<sup>t</sup> or animal HMGR nor has the morphogenic mechanism been described. In *Arabidopsis*, OSER structures induced by the membrane domain of HMGR1S fused to GFP (1S:GFP chimera) also accumulate high amounts of endogenous HMGR and, therefore, have been named ER-HMGR domains [19].

Highly proliferated ER with ordered repetitive patterns was first described in the 1960s, as naturally occurring in diverse cell types from animals and plants [24–30] and as readily developing upon exposure to drugs [31,32]. Since then, diverse forms of hypertrophied ER have been identified in many natural and induced systems and referred to with a variety of terms, such as *cotte de mailles* [33], *paracrystalline arrays* [34], *elaborate rings of granular ER* [35], *double membrane arrays* [36], *tubuloreticular structures* [37], *undulating membranes* [38], *membrane lattice* [39], *stacks of flattened smooth ER* [40], *interlaced smooth surfaced tubules* [41], *compact areas of smooth membranes* [42], *paracrystalline ER* [43], *crystalloid membranes* [44], *organized smooth endoplasmic reticulum* [45] or *cubic membranes* [46]. An exhaustive review [47], with about 200 examples, reported that *cubic membranes* (OSER structures) are broadly distributed in the three eukaryotic kingdoms. These structures are found in numerous cell types under certain physiological conditions or appear in response to stress or disease [47]. However, in most of the aforementioned studies, images were obtained by transmission EM after chemical fixation [47]. Alternative preparation and observation techniques are necessary to further expand our knowledge on OSER ultrastructure.

In this work we first study in more depth the subcellular location of *Arabidopsis* HMGR and, particularly, the HMGR vesicles. We also characterize ER-HMGR domains in *Arabidopsis* and *Nicotiana* cells, focusing on their biogenesis, ultrastructure and dynamism. Our EM analyses uncover differences in OSER ultrastructure because of the fixation method. We find that the ER-HMGR domains are flexible live entities, fully integrated in ER architecture and dynamism.
