Endogenous spartin (SPG20) is recruited to endosomes and lipid droplets and interacts with the ubiquitin E3 ligases AIP4 and AIP5

Abstract

The HSPs (hereditary spastic paraplegias) are genetic conditions in which there is distal degeneration of the longest axons of the corticospinal tract, resulting in spastic paralysis of the legs. The gene encoding spartin is mutated in Troyer syndrome, an HSP in which paralysis is accompanied by additional clinical features. There has been controversy over the subcellular distribution of spartin. We show here that, at steady state, endogenous spartin exists in a cytosolic pool that can be recruited to endosomes and to lipid droplets. Cytosolic endogenous spartin is mono-ubiquitinated and we demonstrate that it interacts via a PPXY motif with the ubiquitin E3 ligases AIP4 [atrophin-interacting protein 4; ITCH (itchy E3 ubiquitin protein ligase homologue] [corrected] and AIP5 (WWP1). Surprisingly, the PPXY motif, AIP4 and AIP5 are not required for spartin's ubiquitination, and so we propose that spartin acts as an adaptor for these proteins. Our results suggest that spartin is involved in diverse cellular functions, which may be of relevance to the complex phenotype seen in Troyer syndrome.

Figure 1. A novel anti-spartin antibody confirms that spartin shows multiple mono-ubiquitination

(A) Western blot (WB) of HeLa, NSC34 and PC12 lysates using α-spartin final bleed (left panel) or pre-immune serum (PI, right panel). (B) HeLa cells transfected with control non-targeting siRNA show a strong ∼80 kDa band on immunoblotting with α-spartin, whereas in cells transfected with each of two siRNA oligonucleotides directed against spartin, or with a pool of the two spartin siRNAs, this band is absent. Actin immunoblotting is shown to verify equal loading. (C) Spartin was immunoprecipitated (IP) from Hela cells by α-spartin, but not by pre-immune serum. (D) Spartin was immunoprecipitated from HeLa cells by α-spartin. Subsequent immunoblotting with FK2 (which recognizes mono- and poly-ubiquitin) detected bands corresponding to the expected size of ubiquitinated forms of spartin, whereas immunoblotting with FK1 (which recognizes only poly-ubiquitin) detected no signal. (E) HeLa cells were transfected with HA-tagged ubiquitin, then immunoprecipitated with α-spartin or with pre-immune serum. Immunoblotting with anti-HA antibody detected bands corresponding to the size of ubiquitinated spartin in the sample immunoprecipitated with α-spartin. (F) Under immunofluorescence confocal microscopy, mock-transfected HeLa cells show a strong cytosolic signal when labelled with α-spartin. However, in cells transfected with an siRNA oligonucleotide directed against spartin, the signal was greatly diminished. Images were obtained using identical confocal microscope settings. Scale bars=10 μm.

Figure 2. Endogenous spartin can be recruited to endosomes

(A) Endogenous spartin was recruited onto Vps4-EQ-positive puncta in HeLa cells transfected with Vps4-EQ–GFP. Arrows indicate individual puncta that show co-localization. The inset shows a magnified image of the boxed area. (B–C) HeLa cells were incubated with EGF (5 ng/ml in medium) on ice for 1 h, then placed into warm serum-free medium to initiate a synchronized pulse of EGFR endocytosis, and processed for immunofluorescence at the indicated times. At 1 min chase, EGFR was predominantly on the plasma membrane and no co-localization with spartin was present. At 30 min chase, spartin showed co-localization with the endocytosed EGFR. Arrows indicate individual puncta that show co-localization. The insets show magnified images of the boxed areas. In (A–C) and in subsequent micrograph panels, right-hand panels show the merged images; the colour of each marker in the merged image is shown by the colour of its lettering in the non-merged panels. Scale bars=10 μm. (D) Graph showing EGFR immunoblot density in mock-transfected or spartin-depleted cells stimulated with 100 ng/ml of EGF for the times indicated (n=4, one-tailed paired t test). The corresponding immunoblots show a representative experiment.

Figure 3. Endogenous spartin can be recruited to lipid droplets

(A) HeLa cells were treated with oleic acid (200 μM for 24 h) and lipid droplets were labelled by incubation with BODIPY 493/503. Cells were then fixed and labelled for spartin. A spartin signal often surrounded the lipid droplets and, where lipid droplets clustered, a honeycomb appearance was seen (see boxed area; inset shows a magnified image of the boxed area). (B) A cell in which lipid droplet formation was induced in the same way as in (A). The cell was labelled with the mitochondrial marker MitoTracker, as well as with α-spartin and BODIPY 493/503. The spartin signal surrounds many of the lipid droplets; an example is indicated by the arrowheads. The spartin label around the lipid droplets shows minimal co-localization with MitoTracker. Scale bars=10 μm.

Figure 4. Spartin interacts with AIP4 and AIP5

(A) Western blotting (WB) against endogenous AIP4 detects an AIP4-sized band after immunoprecipitation (IP) of HeLa cell lysate with α-spartin, but not with pre-immune (PI) serum. (B) Immunoblotting against endogenous AIP5 detects an AIP5-sized band after immunoprecipitation of HeLa cell lysate with α-spartin, but not with pre-immune serum. (C) Endogenous spartin partially co-localizes with endogenous AIP4, in small puncta. Arrows indicate puncta showing co-localization. The inset shows a magnified image of the boxed area. Scale bar=10 μm. (D) Endogenous spartin was immunoprecipitated from HeLa cells transfected with FLAG-tagged wild-type AIP4 or catalytically inactive AIP4 (C803A). Immunoblotting with anti-FLAG demonstrated that wild-type and catalytically inactive AIP4 were co-immunoprecipitated with spartin. Neither spartin nor the AIP4 proteins were immunoprecipitated by pre-immune serum. UT, untransfected cells. (E) Endogenous spartin was immunoprecipitated from HeLa cells transfected with wild-type (wt) or catalytically inactive (C962S; mut.) Myc-tagged Nedd4.2. Immunoblotting with anti-Myc showed that the Nedd4.2 proteins had not been co-immunoprecipitated with spartin. (F) Sequence of the first 210 amino acids of spartin. The MIT (microtubule-interacting and transport) domain is shown in bold, the PPXY motif is boxed. (G) HeLa cells were transfected with FLAG-AIP4 and either wild-type Myc-tagged spartin (WT) or Myc-tagged spartin in which the PPXY motif had been mutated to AAAA. After immunoprecipitation with anti-Myc, FLAG-AIP4 co-immunoprecipitated with WT Myc–spartin, but not Myc–spartinAAAA. (H) HeLa cells were transfected with wild-type Myc–tagged spartin (left panels) or Myc–spartinAAAA (right panels). After immunoprecipitation with anti-Myc, endogenous AIP5 co-immunoprecipited with wild-type Myc-spartin, but not with Myc–spartinAAAA. An additional short exposure is shown so that immunoblotting results for the input lanes can be properly visualized.

Figure 5. PPXY, AIP4 and AIP5 are not required for spartin's ubiquitination

(A) Wild-type (WT) Myc–spartin and Myc–spartinAAAA were immunoprecipitated (IP) from HeLa cells using anti-Myc antibody. Blotting with FK2 (which recognizes poly- and mono-ubiquitin) revealed similar levels of ubiquitination of both forms of spartin. No bands were seen in samples immunoprecipitated with a spurious antibody directed against actin (results not shown). (B) Neither ubiquitination of spartin (as indicated by the density of the higher-molecular-mass ubiquitinated band) nor the concentration of spartin differed between untransfected HeLa cells, HeLa cells transfected with wild-type FLAG-tagged AIP4 (WT AIP4), or with catalytically inactive FLAG–AIP4 (C830A). (C) AIP4 depletion, AIP5 depletion or depletion of both proteins did not affect the concentration of spartin or the strength of the upper ubiquitinated band seen on spartin immunoblotting. In (B) and (C), actin immunoblotting is shown to verify equal loading.