How organelles establish and maintain their morphological and functional identity is a fundamental question in cell biology. The secretory pathway is an excellent system by which to address this question because it comprises numerous organelles that are morphologically and functionally distinct. In most eukaryotes, the first secretory organelle, the endoplasmic reticulum (ER), assumes a characteristic network-like morphology (Baumann and Walz, 2001; Sparkes et al., 2009a). In plants, the Golgi apparatus, which receives secretory materials from the ER for processing and sorting, is composed of dispersed and polarized stacks (Matheson et al., 2006), which move by alternating wiggly and directional motion at a relatively high velocity (Boevink et al., 1998; Nebenfuhr et al., 1999). In highly vacuolated cells, Golgi stacks appear to be attached to the ER surface (Sparkes et al., 2009b). Little is known about how the complex architecture of the ER is formed and maintained, or what role the overall structure plays in ER function. In plant cells, the ER can be split in two interconnected regions: a peripheral network (cortical ER) and an underlying fast-streaming one (inner ER) (Ueda et al., 2011). Live-cell imaging has revealed that the ER is a highly dynamic organelle showing continuous growth, retraction, sliding and fusion of tubules to form strands, cisternae and polygons (Sparkes et al., 2011). ER tubules are pulled and extended from a membrane reservoir by cytoskeletal elements, like microtubules in mammalian cells and characean intermodal cells (Vedrenne and Hauri, 2006; Foissner et al., 2009), or as actin filaments in plant and yeast cells polymerize (Prinz et al., 2000; Brandizzi et al., 2003b). These high-curvature tubules are then stabilized by cytoskeleton-independent mechanisms. Members of a family of membrane curvature-inducing proteins, named reticulons/DP1/Yop1p proteins, have been implicated in such mechanisms (Voeltz et al., 2006; Tolley et al., 2008; Sparkes et al., 2010). Although reticulons and/or DP1/Yop1p appear to be the minimal components required for ER tubule formation in vitro, there may be additional uncharacterized factors that determine the shape of the ER network in vivo (Hu et al., 2009). This is particularly relevant for the dynamic rearrangement of the tubules that follows their stabilization. Dynamin-like proteins such as the metazoan atlastins and the yeast functional ortholog, SEY1, have been implicated in remodeling of the ER, specifically in ER homotypic fusion, as demonstrated by depletion of atlastins and over-expression of dominant-negative mutants that inhibit the formation of tubule interconnections (Hu et al., 2009; Orso et al., 2009). In vitro experiments have shown that Drosophila atlastin drives membrane fusion by bringing the ER membranes into contact through head-to-head interaction of the large GTPase domains and consequent conformational changes of the two juxtaposed atlastin proteins (Moss et al., 2011). It has been suggested that Arabidopsis RHD3 may be a functional ortholog of atlastin and SEY1 (Hu et al., 2009), but several crucial questions remain unanswered. It has been reported that over-expression of RHD3 proteins with mutations in the putative GTPase domain causes formation of unbranched ER and reduced Golgi movement in tobacco leaf epidermal cells (Chen et al., 2011). Because these results were obtained through over-expression of dominant-negative mutants in a heterologous system, it is not yet known to what the extent RHD3 is involved in organization of the ER and Golgi movement in plant cells. Several other important additional questions are also unanswered. For example, the role played by other RHD3 domains in addition to the putative GTPase domain, which is located in the N-terminal region of the protein, is unknown. Also, RHD3 has not been tested to determine whether it is a bona fide GTPase.
Here we report on a mutant, gom8, that was identified through a forward genetics screen of the plant Golgi (Boulaflous et al., 2008). We show that gom8 has reduced Golgi motility and defects in Golgi distribution and ER organization compared to wild-type. The gom8 mutant possesses a proline to serine substitution at position 701 in the anchor domain of the C-terminal region of RHD3. By characterization of gom8 and an RHD3 null mutant, we show that RHD3 plays an important role in organization of the ER, but that between-tubule fusion still occurs in the peripheral ER when the ER is depleted of RHD3, highlighting a dispensable role for RHD3 in tubule fusion. These data also show that integrity of the C-terminal region has a fundamental role in RHD3 function. We further support this conclusion by demonstrating not only that the C-terminal region of RHD3 is essential for ER anchoring, but also that replacement of the RHD3 C-terminal region by the ER anchor domain of PVA12, a type II ER protein, causes loss of RHD3 function.