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

Abstract

The endoplasmic reticulum (ER) network comprises sheets and tubules that are connected by dynamic three-way junctions. Lunapark (Lnp) localizes to and stabilizes ER three-way junctions by antagonizing the small GTPase Atlastin, but how Lnp shapes the ER network is unclear. Here, we used an affinity purification approach and mass spectrometry to identify Lnp as an interacting partner of the ER protein quality control ubiquitin ligase gp78. Accordingly, Lnp purified from mammalian cells has a ubiquitin ligase activity in vitro Intriguingly, biochemical analyses show that this activity can be attributed not only to associated ubiquitin ligase, but also to an intrinsic ubiquitin ligase activity borne by Lnp itself. This activity is contained in the N-terminal 45 amino acids of Lnp although this segment does not share homology to any known ubiquitin ligase motifs. Despite its interaction with gp78, Lnp does not seem to have a broad function in degradation of misfolded ER proteins. On the other hand, the N-terminal ubiquitin ligase-bearing motif is required for the ER three-way junction localization of Lnp. Our study identifies a new type of ubiquitin ligase and reveals a potential link between ubiquitin and ER morphology regulation.

Keywords: Atlastin; ER three-way junction; Lunapark/Lnp; endoplasmic-reticulum-associated protein degradation (ERAD); gp78; misfolded; protein misfolding; ubiquitin; ubiquitin ligase; ubiquitin-conjugating enzyme (E2 enzyme).

FIGURE 1.

Lnp interacts with the ERAD ubiquitin ligase gp78. A, gp78-FLAG pulldown was performed using HEK293T cells transfected with an empty vector (control) or a gp78-FLAG construct. Proteins eluted were analyzed by SDS-PAGE by Coomassie Blue staining. B, co-immunoprecipitation confirms the interaction of Lnp with endogenous gp78. Cells transfected with an empty control vector or a Lnp-FLAG construct were lysed, and proteins immunoprecipited with FLAG beads were analyzed by immunoblotting. C, interaction of Lnp with gp78 in untransfected cells. Whole cell extracts were subject to immunoprecipitation using protein A beads preincubated with the indicated antibodies, and then fractionated into bound and unbound fractions prior to immunoblotting. D, a schematic illustration of Lnp domain structure. PR, proline rich segment; ZF, zinc finger motif. E and F, characterization of the Lnp-gp78 interaction. HEK293T cells transfected with the indicated FLAG-tagged Lnp variants were lysed in a Nonidet P-40-containing lysis buffer. The lysates were subject to immunoprecipitation by FLAG beads. G, a schematic diagram illustrating the interaction of gp78 with Lnp on the ER membrane.

FIGURE 2.

Lnp purified from mammalian cells has a ubiquitin ligase activity. A, Lnp-FLAG purified from HEK293T cells was visualized by Coomassie Blue staining (left panel). The purified Lnp-FLAG (+Lnp) or buffer (−Lnp) was incubated with E1, HA-ubiquitin, or ATP, in the absence (no E2), or presence of UBE2D1 or UBE2G2 for 1 h. The reaction was analyzed by immunoblotting with anti-HA antibody (right panel). B, single round ubiquitin transfer assay was done in the presence or absence of Lnp-FLAG. His-tagged UBE2D1 was charged with HA-tagged ubiquitin and then quenched to block an additional round of ubiquitin charging. The HA-ubiquitin charged E2 was then incubated with untagged ubiquitin in the absence or presence of Lnp-FLAG. Samples were analyzed by immunoblotting under non-reducing or reducing condition. The area indicated by the box under the non-reducing condition is shown with two different exposures. C, purified Lnp-FLAG was further fractionated by a Superdex 200 column to remove gp78. The resulting protein product was analyzed by Coomassie Blue staining and immunoblotting with the indicated antibodies. D, the highly purified Lnp was incubated with E1, UBE2D1, HA-ubiquitin, and ATP to assay the ubiquitin ligase activity. E, Lnp-FLAG purified from either WT or gp78 CRISPR knock-out (ko) cells was incubated with E1, UBE2D1, HA-ubiquitin, and ATP at 37 °C for 1 h. The reaction was analyzed by immunoblotting with anti-HA antibody. Where indicated, Lnp was omitted from the reaction as a negative control. Bottom panels show immunoblotting analysis of cell lysates from control and gp78 knock-out cells (Input). F, mapping of the Lnp domain required for its E3 activity. The indicated Lnp variants were expressed and purified from HEK293T cells. The purified proteins were incubated with E1, UBE2D1, HA-ubiquitin, and ATP at 37 °C for 1 h. The reaction was analyzed by immunoblotting with anti-HA antibody.

FIGURE 3.

Recombinant Lnp purified from E. coli has a ubiquitin ligase activity. A, the indicated amount of His-LnpΔTM and UBE2D1 purified from E. coli were incubated with E1, HA-ubiquitin, and ATP at 37 °C for 1 h. The reaction was analyzed by immunoblotting with anti-HA (top panel) and anti-Lnp (bottom panel) antibodies. B, GST-Lnp(1–45) purified from E. coli was fractionated by size exclusion chromatography and compared with a molecular weight standard. Proteins in the peak fraction was also analyzed by SDS-PAGE and Coomassie Blue staining. C, increased amount of GST-Lnp(1–45) was incubated with E1, UBE2D1, HA-ubiquitin, and ATP at 37 °C for 1 h. The samples were analyzed by immunoblotting with anti-HA antibody. The graph shows the relative intensity of the ubiquitin-positive bands in the gel. Note that the truncated Lnp preferentially synthesizes ubiquitin chains containing 4–5 moieties. D, kinetic analysis of Lnp-mediated ubiquitination. The in vitro ubiquitination reaction was performed in the absence or presence of GST-Lnp(1–45). Samples taken at the indicated time points were analyzed by anti-HA immunoblotting.

FIGURE 4.

Lnp does not play a significant function in ERAD. A, gp78 does not ubiquitinate Lnp in cells. Endogenous Lnp and Ubl4A were immunoprecipitated (IP) under denaturing conditions from cells transiently transfected with HA-ubiquitin together with either a control or a gp78-expressing plasmid. A fraction of the whole cell extract (input) and the precipitated materials were analyzed by immunoblotting with the indicated antibodies. B, Lunapark knockdown does not significantly affect the degradation rate of TCRα-YFP. The degradation rate of the model ERAD substrate TCRα-YFP in control and Lnp knockdown cells was analyzed by a cycloheximide chase experiment using a cell line stably expressing TCRα-YFP. C, the steady state level of the ERAD substrate MHC(1–147) was examined in cells transfected with three control- and two Lnp-shRNA knockdown plasmids. Whole cell extracts prepared at 48 h post-transfection were analyzed by immunoblotting (IB). Asterisk, a nonspecific band. D, the effect of Lnp knockdown and re-expression on the degradation of MHC(1–147). The degradation of MHC(1–147) in control and Lnp knockdown cells was analyzed by a cycloheximide chase experiment using a cell line transiently expressing FLAG-tagged MHC(1–147). Where indicated, Lnp knockdown shRNA was co-transfected with a Lnp-FLAG construct.

FIGURE 5.

The N-terminal domain of Lnp is necessary for its localization to the three-way junction. A, COS7 cells transiently transfected with the indicated FLAG-tagged Lnp constructs together with the ER marker Derlin1-GFP were fixed and stained with anti-FLAG antibody. Scale bars, 5 μm. Two examples of cells expressing wild-type Lnp are shown, representing two distinct ER morphologies seen in these cells. B, a close-up view of cells expressing either Lnp-FLAG or Lnp-FLAG(40–427) is shown in A. Scale bars, 3 μm. C, mCherry-tagged Lnp was transiently expressed together with GFP-Atlastin3 in COS7 cells. Images taken at different time points show strong co-localization of Lnp with Atlastin3. Scale bars, 10 μm.

FIGURE 6.

Live cell imaging analysis of mCherry-tagged Lnp subcellular localization. A, the indicated Lnp variants fused with mCherry were transiently expressed together with mCitrine (mCi)-ER in COS7 cells. 24 h post-transfection, cells were directly imaged by a confocal microscope. Scale bars, 2 μm. B, as in A, except that the indicated Lnp variants were analyzed. Scale bars, 2 μm.