Our results indicate that several mechanisms shape peripheral ER sheets. The most basic and universal mechanism appears to involve the previously identified curvature-stabilizing proteins, the reticulons and DP1/Yop1p. These proteins would not only stabilize the high curvature of narrow tubules, but also the curvature of sheet edges, a mechanism that is sufficient to keep the two membranes of a sheet closely apposed. The reticulons and DP1/Yop1p probably stabilize high curvature by two mechanisms, “hydrophobic insertion/wedging” and “scaffolding” (Shibata et al., 2009
). The conserved segments of these proteins may form a wedge in the lipid bilayer that occupies more space in the cytoplasmic leaflet than in the luminal leaflet. Oligomerization of these proteins may generate scaffolds around curved membranes, which may take the shape of open arcs, given that they can localize to sheet edges. Our theoretical model demonstrates that the reticulons and DP1/Yop1p alone can generate both tubules and sheets, with their abundance determining the ratio of these domains. Consistent with the proposed dual role of the reticulons and DP1/Yop1p in tubule and sheet formation, they localize to both tubules and sheet edges, their depletion leads to increased sheet areas, and their overexpression converts sheets into tubules.
In S. cerevisiae the amount of membrane surface and the abundance of the reticulons and Yop1p appear to be the decisive factors determining the ratio of peripheral ER sheets and tubules. Generating more lipid increases the sheet area, while increasing the abundance of the curvature-stabilizing proteins increases the amount of tubules. The observation of sheets in cells lacking the reticulons and Yop1p may be explained by the presence of other, low abundance curvature-promoting proteins, or by the association of the cortical ER with the plasma membrane. Although we cannot exclude the existence of sheet-promoting proteins in yeast, the current data are consistent with a model in which curvature-stabilizing proteins are the major determinant of peripheral ER morphology.
Our data suggest that in mammalian cells there are several additional factors that determine the morphology of peripheral ER sheets. This includes the coiled-coil membrane protein Climp63, which serves as a luminal spacer. After its depletion, the luminal width of the sheets decreases from ~50 to ~30 nm, a spacing that is also seen in organisms that lack the protein. Climp63 is highly upregulated in mammalian cells with proliferated ER sheets and it induces sheets at the expense of tubules when overexpressed in tissue culture cells. Thus, at high concentrations, Climp63 appears to generate sheets all by itself, and the lack of extensive sheet edges may make the contribution of the curvature-stabilizing proteins less important. However, with luminal spacers alone, one would expect bulging of the sheet edges, in contrast to our observations (), indicating that the curvature-stabilizing proteins may have a role even in cells with proliferated ER sheets. Climp63’s function might be to optimize the size of the luminal space of peripheral ER sheets, such that sufficient luminal chaperones can be accommodated and the sheets are packed into a minimal space.
Our analysis also identified two other coiled-coil membrane proteins, p180 and kinectin, with a potential role in shaping ER sheets. These proteins are enriched in sheets and abundant in cells with proliferated ER sheets. Overexpression of p180 has been reported to induce sheets in S. cerevisiae
and in a monocytic cell line (Becker et al., 1999
; Benyamini et al., 2009
), although in our own experiments and those of others the effects were smaller (Ueno et al.), 2010 and data not shown). The depletion of p180 and kinectin had no effect on ER sheet morphology. Although the precise role of these proteins remains to be established, all coiled-coil membrane proteins could stabilize sheets simply by being excluded from high-curvature regions, as shown by our theoretical considerations. They may be considered as generating an “osmotic pressure,” a force that counteracts the shrinkage of sheet domains. Consistent with experimental observations for Climp63, the coiled-coil proteins are predicted to be in a “tug-of-war” with the reticulons and DP1/Yop1p, with the former shifting the balance towards sheets and the latter towards tubules. In this model it does not actually matter how proteins are excluded from tubules and sheet edges. Given that all identified sheet-promoting proteins contain extended coiled-coil domains, they all have the propensity to oligomerize, which may contribute to their exclusion from high-curvature regions.
The coiled-coil membrane proteins are not essential for sheet formation per se, as is obvious from our observation that their depletion by RNAi does not abolish ER sheets. This suggests that, like in yeast, the reticulons and DP1/Yop1p may provide the basic mechanism by which both sheets and tubules are generated. Consistent with this hypothesis, Climp63, p180, and kinectin are not known in lower organisms, in contrast to the reticulons and DP1/Yop1p, which are present in all eukaryotes.
All sheet-enriched proteins tested, including translocon components and the coiled-coil membrane proteins, appear to be concentrated by membrane-bound polysomes; upon polysome disassembly, all these proteins distribute equally between sheets and tubules throughout the cell. Thus, these proteins must have a direct or indirect affinity for membrane-bound polysomes. Indeed, several of the tested sheet-preferring proteins are known to be associated with membrane-bound translating ribosomes, including components of the Sec61 complex, the TRAP complex, the oligosaccharyl transferase complex, and p180 (Gorlich and Rapoport, 1993
). These proteins stay bound to ribosomes upon detergent solubilization of rough ER membranes, but they can be released from the ribosomes by puromycin/high salt treatment. Climp63 and kinectin are not bound to detergent-solubilized translocons (data not shown), so how they are recruited remains to be clarified.
Our results indicate that ER sheets correspond to rough ER and tubules to smooth ER. We propose that the assembly of translating membrane-bound ribosomes into polysomes concentrates the associated membrane-proteins, including Climp63, p180, and kinectin. Their concentration might facilitate their higher-order oligomerization, which may be required for their exclusion from high-curvature areas and thus for their sheet-promoting function. Once sheets are formed, the membrane binding of polysomes would be facilitated. Polysomes often form spirals that could have an inherent preference for associating with ER sheets (Christensen and Bourne, 1999
); while individual ribosomes or small polysomes can bind to narrow tubules, it is unlikely that each ribosome of a large polysome could be efficiently arranged on a narrow tubule. The binding of large polysomes could therefore be restricted to membrane sheets. The assembly of membrane-bound polysomes would concentrate more coiled-coil membrane proteins, and these in turn would generate more sheet area by the “osmotic effect,” allowing more polysomes to bind, and so on, a mechanism that would ultimately lead to a segregated rough ER domain. This model is consistent with the observation that the disassembly of polysomes or the depletion of Climp63 increases the mobility of translocons in the plane of the membrane (Nikonov et al., 2007
; Nikonov et al., 2002
). It also agrees with our results showing that the disassembly of polysomes leads to the spreading of ER sheets similar to that seen upon depletion of the sheet-promoting proteins. Our model explains the classic observation that in many cells membrane-bound ribosomes are not randomly distributed throughout the ER, but rather concentrated in a separate membrane domain, the rough ER. An active sorting of proteins into the rough ER is consistent with previous cell fractionation experiments, which demonstrated that general ER proteins indiscriminately distribute throughout the ER, whereas translocon-associated proteins are enriched in the rough ER (Hinman and Phillips, 1970
; Kreibich et al., 1978
; Vogel et al., 1990
The nuclear envelope is a prominent ER domain whose structure is determined independently of the peripheral ER. Although the reticulons have been implicated in the assembly of the nuclear envelope and in the insertion of nuclear pores (Anderson and Hetzer, 2008
; Dawson et al., 2009
), they are nearly absent from the nuclear envelope, and their depletion or overexpression has no significant effect on this domain’s morphology. Similarly, DP1/Yop1p or the coiled-coil membrane proteins Climp63, p180, and kinectin are also nearly absent from the nuclear envelope and have no obvious effect on its structure. Interestingly, TRAPα was also depleted from the nuclear envelope, raising the possibility that translocons are preferentially located in peripheral ER sheets. Thus, distinct mechanisms may determine the formation and function of the sheet-like domains of the nuclear envelope and peripheral ER.
In summary, our results lead to a simple model, according to which the basic morphological elements of the peripheral ER, the tubules and sheets, are generated by the curvature-stabilizing proteins. Superimposed on this mechanism, membrane-bound polysomes and associated coiled-coil membrane proteins may cooperate to form segregated rough ER sheets in mammalian cells, domains that are functionally specialized in protein translocation. Other factors probably contribute to the morphology of the peripheral ER. Microtubules keep the mammalian ER under tension and stabilize membrane tubules, but they could also potentially form an additional scaffold that stabilizes sheets, as suggested by the fact that both Climp63 and p180 are microtubule-binding proteins (Klopfenstein et al., 1998
; Ogawa-Goto et al., 2007
). It will be interesting to elucidate how these factors collaborate with the identified membrane-shaping principles.