Interactome of the plant-specific ESCRT-III component AtVPS2.2 in Arabidopsis thaliana

Abstract

The endosomal sorting complexes required for transport (ESCRT) guides transmembrane proteins to domains that bud away from the cytoplasm. The ESCRT machinery consists of four complexes. ESCRT complexes 0-II are important for cargo recognition and concentration via ubiquitin binding. Most of the membrane bending function is mediated by the large multimeric ESCRT-III complex and associated proteins. Here we present the first in vivo proteome analysis of a member of the ESCRT-III complex which is unique to the plant kingdom. We show with LC-MS/MS, yeast-two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) that coimmunoprecipitated proteins from Arabidopsis thaliana roots expressing a functional GFP-tagged VACUOLAR PROTEIN SORTING 2.2 (AtVPS2.2) protein are members of the ESCRT-III complex and associated proteins. Therefore we propose that at least in plants the large ESCRT-III membrane scaffolding complex consists of a mixture of SNF7, VPS2 and the associated VPS46 and VPS60 proteins. Apart from transmembrane proteins, numerous membrane-associated but also nuclear and extracellular proteins have been identified, indicating that AtVPS2.2 might be involved in processes beyond the classical ESCRT role. This study is the first in vivo proteome analysis with a tagged ESCRT-III component demonstrating the feasibility of this approach and provides numerous starting points for the investigation of the biological process in which AtVPS2.2 is involved.

Figure 1

AtVPS2.2-GFP complements vps2.2–3 phenotypes and is predominantly expressed in root meristems. (A) Wildtype and vps2.2–3 mutants expressing AtVPS2.2-GFP develop significantly longer roots than untransformed vps2.2–3, substantiating the functionality of the construct (p_ab < 4.4 × 10^–5 for all comparison to vps2.2–3). Yet all transformants develop shorter roots than untransformed wildtype seedlings (p_bc < 0.004). Root lengths were determined of at least 20 seedlings 7 days after germination on MS medium supplemented with 4.5% sucrose. The significance was calculated by students t-tests. (B) Stability and prevalent root expression of the AtVPS2.2-GFP fusion protein and the specificity of the GFP-antibody was determined by Western blot analyses loading 75 μg total proteins of wildtype root (wt), of seedlings expressing an unstable GFP-fusion protein (GFP-control), roots and shoot extracts of wildtype seedlings expressing AtVPS2.2-GFP. While in the unstable GFP-control extract a degradation product is detectable (star, 26 kD) the AtVPS2.2-GFP fusions (50 kD) are stable. (C) Roots of 14 days old seedlings expressing AtVPS2.2-GFP. AtVPS2.2-GFP is predominantly expressed in root meristems.

Figure 2

Strategy to purify and identify AtVPS2.2-GFP interacting proteins. (A) Roots were separated from seedlings, cushed in liquid nitrogen, lysed with cracking buffer on ice and centrifuged to separate cell debris. Anti-GFP μMACS MicroBeads were added to the supernatant, mixed by rotating and after incubation on ice applied onto μ Columns. The eluted proteins were separated on a denaturing 12% (w/v) PAGE and visualized with a LC–MS/MS compatible silver staining protocol. Lanes were excised into up to 29 fractions and subjected to nanoelectrospray LC–MS/MS sequencing. (B) Example of a silver stained PAGE showing the eluates from extracts of control roots (wildtype), transgenes expressing AtVPS2.2-GFP and the eluted beads. Indicated are proteins which have been specifically enriched by this method.

Figure 3

In vivo interaction studies of AtVPS2.2 with known and novel interacting partners of the ESCRT-III complex. (A) Yeast two hybrid analyses showing growth assays for homo- and heterodimerization and the autoactivation of the AtVPS60.1 DNA-binding domain fusion. The identity of the cotransformants is indicated on the left, the selective media above and the strength of the interactions on the right. Shown are spotted undiluted and 1:10 diluted cultures. (B) BiFC interactions in Arabidopsis protoplasts demonstrating homo- and heterodimerizations of AtVPS2.2 with AtVPS2.1, AtSNF7.1, AtVPS46.1 and AtVPS60.1. The interactions label vesicles in the cytoplasm and at the plasma membrane.

Figure 4

Subcellular localization of AtVPS2.2 interacting proteins. Venn diagrams of the overlap between AtVPS2.2-GFP interactors and (phosphor-) proteins that have been isolated from membranes and vacuoles. The colored AGI codes of the proteins correspond to the overlapping sectors. The first authors of the publications used for comparison are also color coded.

Figure 5

AtVPS2.2-GFP localizes to distinct region of the plasma membrane, in cytoplasmic vesicles separated for the trans-Golgi network (TGN) marker VHAa1 and in nuclei of root meristems. (A) Root tips of seedlings expressing AtVPS2.2-GFP, (B) root meristems of seedlings expressing AtVPS2.2-GFP and the TNG marker VHAa1-mRFP, (C) close up of the marked area in B demonstrating that AtVPS2.2-GFP localizes strongly to the transverse cell borders, to vesicles of different sizes in the cytoplasm and diffusely in the nucleus. (D) Root area at the upper end of the meristem with diffuse nuclear AtVPS2.2-GFP localization. (E) Same root area as in D but labeled with the fluorescent DNA stain, DAPI. (F) Overlay of D and E. (G) Venn diagram of the overlap between AtVPS2.2-GFP interactors and proteins that have been isolated from and annotated to the nucleus. The colored AGI codes of the proteins correspond to the overlapping sectors. The first authors of the publications used for comparison are also color coded.

Figure 6

Atypical subcellular localization of AtVPS2.2 interacting proteins. Venn diagrams of the overlap between AtVPS2.2-GFP interactors and proteins that have been isolated from and annotated to the apoplast, cell wall and phloem sap. The colored AGI codes of the proteins correspond to the overlapping sectors. The first authors of the publications used for comparison are also color coded.

Figure 7

Model how ESCRT-III might mediate vesicle budding and fission in conjunction with dynamins and PLDα. Several dynamins assemble and interact with the ESCRT-III subunit AtVPS2.2 on membrane domains on the inner side of the neck which is acidified by PLDα. This association supports membrane narrowing and lipid rearrangements at the neck of budding vesicles away from the cytoplasm so that membrane fission and fusion occurs. Since AtVPS2.2-GFP interacts also with other ESCRT-III components we propose that at least in plants the large membrane scaffolding complex consists of a mixture of AtSNF7, AtVPS2 and the associated AtVPS46 and AtVPS60 proteins. Dark green, AtVPS20; middle green, AtSNF7; blue, AtVPS2; dark blue, AtVPS24; light yellow, AtVPS46; light gray, AtVPS60; dark gray, PLDα; yellow to orange, dynamins; gray, neutral lipids; red, acidified lipids. Arrows indicate the forces that upon constriction of ESCRT-III and dynamins mediate membrane fission and fusion.