Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network

Abstract

In higher eukaryotes, the endoplasmic reticulum (ER) contains a network of membrane tubules, which transitions into sheets during mitosis. Network formation involves curvature-stabilizing proteins, including the reticulons (Rtns), as well as the membrane-fusing GTPase atlastin (ATL) and the lunapark protein (Lnp). Here, we have analyzed how these proteins cooperate. ATL is needed to not only form, but also maintain, the ER network. Maintenance requires a balance between ATL and Rtn, as too little ATL activity or too high Rtn4a concentrations cause ER fragmentation. Lnp only affects the abundance of three-way junctions and tubules. We suggest a model in which ATL-mediated fusion counteracts the instability of free tubule ends. ATL tethers and fuses tubules stabilized by the Rtns, and transiently sits in newly formed three-way junctions. Lnp subsequently moves into the junctional sheets and forms oligomers. Lnp is inactivated by mitotic phosphorylation, which contributes to the tubule-to-sheet conversion of the ER.DOI: http://dx.doi.org/10.7554/eLife.18605.001

Arabidopsis Lunapark proteins are involved in ER cisternae formation

“…Yeast and mammalian Lnp proteins preferentially localize to the three‐way junctions of the ER network and it has been suggested that in these organisms Lnp proteins are involved in stabilizing these intersections (Chen et al ., 2012, 2015; Wang et al ., 2016). Although our results showed that AtLNP1 preferentially localizes to ER cisternae rather than junctions, we wanted to determine if AtLNP1 still has a comparable function in stabilizing the overall ER network.…”

“…This is in contrast to the subcellular localization described for yeast and mammalian Lnp proteins which is rather specific to three‐way junctions; yeast and mammalian Lnp proteins label c . 50% of three‐way junctions, resulting in their stabilization (Chen et al ., 2012; Shemesh et al ., 2014; Wang et al ., 2016). Both AtLNP1 and its animal counterpart can also label ER tubules at higher protein expression levels (Shemesh et al ., 2014; Wang et al ., 2016).…”

“…Mammalian Lnp1 is dependent on N ‐myristoylation for its localization to ER junctions and morphogenic activity. The absence of this motif in yeast Lnp1p could indicate that LNP proteins in higher organisms have evolved an additional degree of functionality and/or regulation via post‐translational lipid modifications (Moriya et al ., 2013; Wang et al ., 2016; Turnbull & Hemsley, 2017). …”

“…Yeast and mammalian LNP proteins preferentially localize to ER three‐way junctions in vivo and, although they are not required for ER network formation, it has been suggested that LNP acts to stabilize these intersections, potentially acting as temporary scaffold in nascent junctions (Chen et al ., 2012, 2015; Wang et al ., 2016). Immunofluorescence staining of mammalian COS‐7 cells with an anti‐mLnp1 antibody showed that only around half of all three‐way junctions contained mLnp1 but that mLnp1 acquisition was associated with enhanced junction survival probability and reduced junction mobility and ring closure (Chen et al ., 2015).…”

“…However, more recently Wang et al . (2016) reported that in mammalian cells stably expressing both Lnp‐mCherry and GFP‐ATL‐3, ATL was in close proximity to Lnp‐mCherry in the peripheral ER network but, crucially, the two proteins did not precisely colocalize and no interaction was found in reciprocal pull‐down experiments.…”