The ER performs a variety of functions in cells, including but not limited to lipid synthesis, Ca2+
handling, and protein translocation and secretion [1
]. To perform its many functions, the eukaryotic ER has perhaps the most complicated structure of any organelle. Some ER domains are obvious and can be distinguished by their shapes using fluorescence microscopy. These include the nuclear envelope (NE), and cytoplasmic cisternae and tubules that form the interconnected peripheral ER ().
ER morphological domains in mammalian cells and yeast
The NE is a distinct domain of the ER made up of two large, flat membrane bilayers, the inner and outer nuclear membranes (INM and ONM). The INM and ONM are separated by the perinuclear space (PNS), but are connected to each other at nuclear pores [2
]. The peripheral ER branches out of the ONM as an extensive network of cisternae and tubules and extends into the cytoplasm all the way to the plasma membrane (PM). The lumen of the peripheral ER is continuous with the lumen of the PNS. What discriminates ER tubules from cisternae is their very different shapes. ER tubules have high membrane curvature at their cross-section, whereas cisternae are comprised of extended regions of parallel flat membrane bilayers that are stacked over each other with regions of membrane curvature found only at their edges. There are, however, similarities between ER tubules and cisternae; specifically, the diameter of an ER tubule is similar to the thickness of an ER cisterna (~38 nm vs. ~36 nm, respectively in yeast) [3
Multiple regions of high membrane curvature in the ER are stabilized by the reticulon and DP1/Yop1 (also called REEP) family of proteins; these regions include the tubules and edges of cisternae and fenestra (Box 1
]. The abundance of reticulon/DP1/Yop1 proteins regulates the abundance of high curvature regions within the peripheral ER [4
]. It is not currently known, however, how the expression level or activity of individual members of this family of membrane-shaping proteins is regulated to fine-tune ER shape. In mammalian cells, there are multiple spliced isoforms of four reticulon family members; each contains a reticulon homology domain, but the members have unique domains, interactions, and expression patterns that likely allow for particular ER tubule arrangements and might confer additional functions (for review, see [10
]). Additionally, the prevalence of cisternae versus tubules can vary, suggesting that ER shape can be adapted to a cell’s ER functional requirements [11
]. For example, secretory cells, such as those in the pancreas, must translocate and secrete a large number of proteins and these cells contain abundant cisternae that are densely studded with ribosomes. In contrast, the ER in muscle cells must rapidly regulate Ca2+
levels during contraction and these cells are enriched in ER tubules that are devoid of ribosomes. In yeast cells, which contain both ER cisternae and tubules, cisternae have a higher ribosome density than tubules (~600–1100/μm2
, respectively) [4
]. These correlations suggest that either cisternae are better suited for ribosome binding and/or ribosome binding stabilizes cisternal ER structure. In animal cells, proteins associated with the translocation machinery are also enriched in cisternae [8
]. Furthermore, changing ER ribosome density can alter ER shape, as treatment of animal cells with puromycin causes ribosome displacement followed by loss of ER cisternae and gain of ER tubules [12
]. These data suggest that ER cisternae may be the preferred site of protein translocation.
Box 1. How ER tubules and sheets get their shape
The peripheral ER in most cells contains a mixture of interconnected membrane tubules and cisternae. The relative amount of tubules versus cisternae depends to a large extent on the proteins that regulate ER membrane curvature, the reticulons and DP1/Yop1. These proteins are conserved integral membrane proteins that can be found in all eukaryotes. They partition exclusively into regions of the peripheral ER that have high membrane curvature, which includes the edges of cisternae as well as tubules ([5
] and ). Initial work both in vitro
and in vivo
identified these proteins as the major factors necessary for organizing the ER membrane bilayer into the shape of a tubule [5
], but they also organize membrane curvature at the edges of cisternae and fenestrations [4
Box 1, Fig. I
Models of how reticulon proteins shape regions of high membrane curvature in the peripheral ER
Reticulons contain a reticulon homology domain (RHD) comprised of two long (30–35 amino acid) transmembrane domains, separated by a soluble linker. These transmembrane domains insert as hairpins in the cytoplasmic leaflet of the ER membrane bilayer ([5
] and ). The short length of these hairpin domains is required in animal cells for: i) partitioning into regions of membrane curvature and ii) generating membrane curvature [65
]. Reticulon proteins also oligomerize into immobile higher-ordered structures in the ER membrane, a requirement for proper tubule formation [67
]. Therefore, a reasonable model of reticulon function is that they form hairpin structures that oligomerize in the outer leaflet of the ER membrane to increase outer leaflet area relative to inner leaflet area, thus generating membrane curvature by altering bilayer symmetry.
In contrast, relatively little is understood about ER cisternae. These domains are comprised of flat areas of ER membrane that are evenly spaced around the ER lumen. At the edges the flattened membrane curves around, forming a pocket. Climp63 partitions into ER cisternae and its overexpression propagates the formation of cisternal ER at the expense of tubules [8
]. When Climp63 is depleted, cisternae persist, but their intra-lumenal spacing is altered [8
]. These data suggest that while Climp63 is not required for cisternae formation, it may form intra-lumenal linker complexes that regulate cisternal dimensions.
A recent paper showed that an additional function of ER cisternae is in the unfolded protein response (UPR). This study demonstrated that in yeast, ER stress-induced UPR leads to membrane expansion of the ER into cisternae [13
]. The cisternal shape is not what allows the stress response, however, because conversion of sheets to tubules does not inhibit stress alleviation [13
]. The authors proposed a model where the important feature of membrane expansion is increased ER volume to allow for additional handling of misfolded proteins. Recent work has shown that the volume-to-surface area ratio of such cisternae in wild type cells exceeds that of ER tubules [4
], indicating that the cisternae formed upon ER stress are the most favorable conformation to meet the need for increased ER volume.
Whether ER tubules are enriched for other ER functions remains to be determined. For a long time, it was thought that because ER cisternae are the preferred site of ER protein translocation, then ER tubules must perform the lipid synthesis and Ca2+
handling functions of the ER. There is no evidence that either process localizes preferentially to ER tubules, however. ER tubules might be the preferred site for ER vesicle budding because the high curvature of ER tubules could be better suited for the formation of ER-to-Golgi transport vesicles (COPII) at ER exit sites (ERES). Markers for ERES localize to ER tubules [14
], although it is not known if these markers are excluded from ER cisternae. It has also been difficult to demonstrate whether other specific ER functions preferentially localize to either cisternae or tubules. Tubules in the ER might simply provide a useful architecture to allow distribution throughout the cytoplasm as a continuous network without disrupting general diffusion and trafficking. In addition to cisternae and tubules, other peripheral ER subdomains defined by their unique functions and locations include the ERES as well as regions that contact the PM, organelles, and the cytoskeleton. What makes these domains difficult to study is that they can only be visualized by electron microscopy or by using multiple fluorescent markers and confocal microscopy in well-resolved cells. We will discuss ER domains that form contact sites with other organelles later in this review.