Lunapark stabilizes nascent three-way junctions in the endoplasmic reticulum

Abstract

The endoplasmic reticulum (ER) consists of a polygonal network of sheets and tubules interconnected by three-way junctions. This network undergoes continual remodeling through competing processes: the branching and fusion of tubules forms new three-way junctions and new polygons, and junction sliding and ring closure leads to polygon loss. However, little is known about the machinery required to generate and maintain junctions. We previously reported that yeast Lnp1 localizes to ER junctions, and that loss of Lnp1 leads to a collapsed, densely reticulated ER network. In mammalian cells, only approximately half the junctions contain Lnp1. Here we use live cell imaging to show that mammalian Lnp1 (mLnp1) affects ER junction mobility and hence network dynamics. Three-way junctions with mLnp1 are less mobile than junctions without mLnp1. Newly formed junctions that acquire mLnp1 remain stable within the ER network, whereas nascent junctions that fail to acquire mLnp1 undergo rapid ring closure. These findings imply that mLnp1 plays a key role in stabilizing nascent three-way ER junctions.

Lunapark Is a Component of a Ubiquitin Ligase Complex Localized to the Endoplasmic Reticulum Three-way Junctions

“…It is conceivable that the two ligases may function together to regulate a certain aspect of ER network construction by mediating ubiquitination of an unknown factor. Although our study does not reveal an apparent abnormality in ER morphology in gp78 CRISPR knock-out cells, it is noteworthy that the ER morphology change observed in Lnp knockdown mammalian cells is subtle (8). Thus, more sensitive assays are necessary to reveal the function of the Lnp-gp78 complex in ER morphology regulation.…”

“…The Lnp homologous protein Lnp1p in budding yeast acts in conjunction with Rtn1p to antagonize Sey1p, the yeast homologue of Atlastin (9). Recently, it was reported that the localization of Lnp to the ER three-way junctions plays a role in stabilizing the polygonal network of the dynamic ER structure (8). Here, we identify Lnp as an interacting protein for the ER-anchored ubiquitin ligase gp78, an enzyme with a well established function in ER-associated protein quality control.…”

“…Concurrently, a small amount of di-ubiquitin molecules consisting of a HA-tagged ubiquitin and an untagged ubiquitin was formed (Fig. 2B, lanes [5][6][7][8]. This species was not sensitive to the reducing agent ␤-mercaptoethanol (lanes 9 -16), suggesting that the ubiquitin moieties are linked by the iso-peptide bond rather than thioester bond.…”

Advances in Plant ER Architecture and Dynamics

“…In metazoans and yeast, it has been shown that lunapark (Lnp1), a two-transmembrane domain protein, is required for ER shaping. In particular, Lnp1 has been implicated in contributing to the tubule-to-sheet conversion, most likely by stabilizing the three-way ER junctions (Chen et al, 2015;Wang S. et al, 2016). A functional ortholog of Lnp1 has yet to be identified in Arabidopsis, and it cannot be yet excluded that other proteins may have analogous functions to Lnp1 or that the stabilization of the plant ER junctions depends on different mechanisms compared with nonplant cell systems.…”

Signaling Microdomains within the Myoendothelial Junction

“…This translates to higher organisms as the same researchers determined that Lnp1 helps maintain ER structure in mammalian cells. Only 50% of junctions expressed Lnp1, but those that had Lnp1 were less dynamic 90 , indicating that junctions can form without Lnp1 but that they are less stable.…”