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The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo

An Author Correction to this article was published on 12 January 2023

This article has been updated

Abstract

Endocytosis controls the perception of stimuli by modulating protein abundance at the plasma membrane. In plants, clathrin-mediated endocytosis is the most prominent internalization pathway and relies on two multimeric adaptor complexes, the AP-2 and the TPLATE complex (TPC). Ubiquitination is a well-established modification triggering endocytosis of cargo proteins, but how this modification is recognized to initiate the endocytic event remains elusive. Here we show that TASH3, one of the large subunits of TPC, recognizes ubiquitinated cargo at the plasma membrane via its SH3 domain-containing appendage. TASH3 lacking this evolutionary specific appendage modification allows TPC formation but the plants show severely reduced endocytic densities, which correlates with reduced endocytic flux. Moreover, comparative plasma membrane proteomics identified differential accumulation of multiple ubiquitinated cargo proteins for which we confirm altered trafficking. Our findings position TPC as a key player for ubiquitinated cargo internalization, allowing future identification of target proteins under specific stress conditions.

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Fig. 1: The viable nosh mutant in the TPC subunit TASH3 has reduced endocytic capacity.
Fig. 2: The mutant nosh is defective in endocytosis.
Fig. 3: The association between the hexameric core an the AtEH/PAn1 subunits is destabilized in nosh.
Fig. 4: The SH3 domain of TASH3 binds poly-ubiquitin chains.
Fig. 5: Ubiquitinated PM proteins accumulate in nosh seedlings.
Fig. 6: The SH3 domain of TASH3 recognizes ubiquitinated cargo.

Data availability

All materials are available from the corresponding authors upon request. All data generated or analysed during this study are included in this article and its Extended Data) and/or in public repositories. The raw mass spectrometry data and MaxQuant result files have been deposited to the ProteomeXchange Consortium via PRIDE (PXD035444). The script for comparative quantification of fluorescent signal at PM versus cytoplasm is available for download (https://github.com/pegro-psb/Cyto-PM-signal-quantification). Araport11plus database consisting of the Araport11_genes.2016.06.pep.fasta downloaded from arabidopsis.org was used for MS analysis. Source data are provided with this paper.

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Acknowledgements

We would like to thank T. Xu (FAFU-UCR, Fuzhou) and J. Friml (IST Austria) for sharing TMK1 seeds, G. Vert (CNRS, Toulouse) for sharing BRI1 and BRI1-25KR seeds, E. Russinova for providing 35S::eGFP seeds and R. Owens (OPPF, Research Complex, Harwell) for sharing an aliquot of the HIS-GFP vector. We express our gratitude to the VIB proteomics core facility for the help and expertise with running all MS experiments and the VIB protein core facility for the help and expertise with protein purifications. This work was supported by the European Research Council Grant T-REX 682436 (D.V.D.); the Research Foundation–Flanders (FWO) 1226420N (P.G.), 12S7222N (J.M.D.), 1124621N (A.D.M.) and G017919N (M.K.); the Czech Science Foundation 22-35680 M (R.P.); and the China Scholarship Council Grant 201906760018 (Q.J.).

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Contributions

P.G. and D.V.D. designed the research and wrote the manuscript. P.G. performed most of the experiments. A.D.M. and K.Y. cloned and purified the SH3 domain. A.D.M. performed the SH3 ubiquitin-binding assay, partitioning assay and PM fraction analysis. J.M.D. and M.V. helped with cloning. R.P. performed in silico docking and phylogenetic analysis. D.E. performed MS analysis. M.K. raised and characterized the AtEH1/Pan1 antibody. J.N. prepared samples for AP–MS analysis. Q.J. and B.P. created the script for measuring fluorescent signal on confocal images. E.M. performed tip-tracking experiments. B.D.R., G.D.J., D.V.D. and P.G. were responsible for experimental design and research supervision. All authors contributed to finalizing the text.

Corresponding authors

Correspondence to Peter Grones or Daniël Van Damme.

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Nature Plants thanks Takashi Ueda, Jian Feng Ma and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Phenotypical assessment of the nosh truncation.

(a) Quantification of genotyping PCR reactions on the F1 progeny of tash3-1 (), tash3-2 () and nosh () backcrossed into Col-0 () to evaluate the transfer of the T-DNA allele via the pollen. The male sterility of tash3-1 and tash3-2 mutants prevents transfer of the T-DNA insertion to the next generation (only WT band amplified). nosh mutants produce viable pollen and can transfer the T-DNA insertion to the next generation (WT band and T-DNA band amplified). (b) Amino acid sequence of the TASH3 C terminus and predicted amino acid sequence of the nosh C terminus, based on the sequencing of the T-DNA insertion site. The body part sequence of TASH3 is marked in blue and the SH3 domain sequence is depicted in green. The sequence that is altered in nosh is underlined. (c) Representative examples of 5- week-old Col-0 and nosh plants grown under long-day conditions (16 h light/8 h dark). Under these conditions, nosh exhibits predominantly delayed flowering. (d) Representative examples of 8-week-old Col-0 and nosh plants grown at 12 h light/12 h dark conditions. Under these conditions, nosh exhibits reduced rosette growth and early senescence.

Extended Data Fig. 2 Complementation of nosh with full-length TASH3-GFP restores its endocytic defects.

(a, b) Representative single-slice spinning-disk images and box plot graphs of endocytic foci densities in epidermal dark-grown hypocotyl cells of TPLATE-GFP (tplate), TASH3-GFP (tash3-1) and two independent TASH3-GFP expressing lines in the nosh background. The densities of endocytic foci in both complemented nosh mutants are similar to the values in TPLATE-GFP (tplate) and to those in the complemented tash3-1 mutant allele (TASH3-GFP in tash3-1). Numbers of quantified cells (2 cells per seedling) are indicated. The top and bottom lines of box plots represent 25th and 75th percentiles, the centre line is the median and whiskers are the full data range. The statistical test used was a two-sided Wilcoxon-signed rank test by comparing mutants to wild type. No adjustment for multiple comparisons was performed. Scale bar = 5 µm. (c, d) Representative kymographs and violin plot graphs of the life-time measurements from the spinning-disk time lapses from panel a. Analogous life-time distributions of endocytic events were observed for TASH3-GFP in nosh as for TPLATE-GFP (tplate) and TASH3-GFP (tash3-1). The number of events analysed for each independent line is indicated at the bottom of each graph. At least 12 movies from 6 seedlings were imaged and analysed for each independent transgenic line. The widest part of the violin plot represents the highest point density, whereas the top and bottom are the maximum and minimum data respectively. Red circles represent the mean and the red line represents the standard deviation. The statistical test used was a two-sided Wilcoxon-signed rank test by comparing mutants to wild type. No adjustment for multiple comparisons was performed. Scale bar = 50 µm. n.s. = not significant.

Source data

Extended Data Fig. 3 Comparative interactomics and AtEH1/Pan1 antibody specification.

(a) Graph depicting the normalized peptide intensities of TPC subunits obtained from MS analysis. For each TPC subunit, the intensities of only those peptides that were present in all experiments (for both baits and in all replicas) were averaged and normalized to the values of the corresponding bait protein. Error bars correspond to ± SD and are based on three technical repeats. The results show that nosh does not affect the hexameric TPC formation, but that the association with the AtEH/Pan1 proteins is weakened. (b) Coomassie-stained gel of the obtained SEC fractions and a quality control HPLC analysis performed using a Superdex 200 increase 10/300 for the batch of recombinant AtEH1 C-term fragment used for rabbit immunization. Fractions 18–23 were pooled to immunize (marked in green). (c) Coomassie-stained gel of the obtained SEC fractions and a quality control HPLC analysis performed using a Superdex 200 increase 10/300 for the batch used for antibody purification. Fractions 25–29 were pooled for purification (marked in green). (d) Stain-free gel and blot for testing AtEH1/Pan1 antibody specificity. In the Col-0 sample only the native AtEH1/Pan1 band is prominently observed (marked with a black arrowhead), while in the pH3.3::AtEH1/Pan1-mRuby3 (Col-0) sample, both the native and the transgene fusion protein (marked with a white arrowhead) can be observed. Two homozygous pH3.3::AtEH1/Pan1-mRuby3 (ateh1/pan1 1-2 −/−) lines show only the transgene fusion protein (marked with white arrowhead).

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Extended Data Fig. 4 A TSAUCER-like variant is produced in nosh.

(a) TASH3 protein structure with annotated peptides identified by MS analysis. Peptides marked in green were present in both samples, while peptides in magenta were missing in the TPLATE-GFP (nosh/tplate) sample. (b) Amino acid sequence alignment of TASH3 (Arabidopsis) and TSAUCER (Dictyostelium) showing that the linker and SH3 domain are missing at the C terminus of the TSAUCER protein (outlined in red). (c) Phylogenetic tree representing the maximum likelihood phylogeny of TASH3. Numbers at nodes correspond to the approximate likelihood ratio test with Shimodaira–Hasegawa-like support. Red circles mark the presence of SH3 domain(s).

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Extended Data Fig. 5 TASH3 interacts with AtEH/Pan1 subunits through its body and not via its SH3 domain.

(a) Schematic representation of AtEH1/Pan1 and AtEH2/Pan1 proteins with PxxP motifs indicated by orange lines. (b–d) Maximal intensity projection of representative Z-stacks of transiently expressed TASH3-GFP, TASH3_body-GFP and mCherry-TASH3_linker_SH3 in epidermal N. benthamiana cells, respectively. (e, f) Maximal intensity projection of representative Z-stacks of TASH3-GFP recruitment to AtEH1/Pan1-mCherry and AtEH2/Pan1-mCherry positive foci upon transient co-expression in epidermal N. benthamiana cells. (g, h) Maximal intensity projection of representative Z-stacks of TASH3_body-GFP recruitment to AtEH1/Pan1-mCherry and AtEH2/Pan1-mCherry positive foci upon transient co-expression in epidermal N. benthamiana cells. (i, j) Representative Z-stacks showing that mCherry-TASH3_linker_SH3 is not recruited to AtEH1/Pan1-GFP or AtEH2/Pan1-GFP positive foci upon transient co-expression in epidermal N. benthamiana cells. White arrowheads indicate colocalization. Scale bar = 20 µm. (k, l) Box plot graphs of the particle/cytoplasm intensity of TASH3, TASH3_body and TASH3_linker_SH3 upon transient co-expression of AtEH1/Pan1 and AtEH2/Pan1 in N. benthamiana. The number of Z-stacks analysed for each combination is indicated at the bottom of each graph. The top and bottom lines of box plots represent 25th and 75th percentiles, the centre line is the median and whiskers are the full data range. Letters represent a two-sided mixed linear model statistic used to determine the difference between samples. No adjustment for multiple comparisons was performed. One outlier is marked with a red asterix. TASH3 and TASH3_body are recruited to AtEH/Pan1, whereas TASH3_linker_SH3 is not.

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Extended Data Fig. 6 Modelling of protein–protein interaction between TASH3_SH3 and Ubiquitin10 or ATG8.

Three best-scoring models from two different modelling algorithms (HDOCK and AlphaFold2) show almost identical binding interfaces between TASH3_SH3 and Ubiquitin10. In contrast, modelling ATG8a-TASH3_SH3 did not result in a single orientation of ATG8a towards the TASH3_SH3 domain as shown for three best-scoring models calculated by AlphaFold2 or HDOCK.

Extended Data Fig. 7 Recombinant TASH3_SH3 domain purification.

(a) Protein sequence of the construct used for recombinant TASH3_SH3 production. (b) Predicted structural model of amino acids 1136–1198 of TASH3 by Alphafold2. (cf) Purification of recombinant HIS-TEV-SH3 domain (TASH3). The collected fractions are indicated by a dotted line. (c) Immobilized metal affinity chromatogram of the first step in the purification. (d) Size exclusion chromatogram of the second step in the purification. The fractions collected in the first step were loaded on a HiLoad® 16/600 superdex®75 pg column. (e) Reverse Immobilized metal affinity chromatogram of the third step in the purification. HIS-TEV-SH3 collected in the second step was subjected to overnight TEV cleavage with HIS-TEV protease and loaded on a HisprepTM Fast Flow 1 ml column (HIS-TEV was removed). (f) Size exclusion chromatogram of the final step in the purification. The fractions collected in the third step were loaded on a HiLoad® 16/600 superdex®75 pg column. (g) Coomassie-stained SDS–PAGE gel of the different steps of the TASH3_SH3 domain purification process. IMAC – collected fractions from c, SEC-I – collected fractions from d, TEV cleavage - the cleavage of the HIS-TEV from the recombinant protein using a HIS-TEV protease, R.IMAC - the collected fractions from e, SEC-II – the collected fractions from f. HIS-TEV-SH3: 8.8 kDa, SH3: 7.0 kDa, HIS-TEV protease: 28 kDa. (h) Western blot detection using an anti-5xHIS antibody on the collected fractions of the second step of purification (SEC-II), showing that the collected fractions contain HIS-TEV-SH3. (i) Coomassie-stained SDS–PAGE gel showing the coupling efficiency of HIS-HRV3C-GFP and TASH3_SH3 on PierceTM NHS-Activated agarose beads. Covalent coupling of the recombinant protein to the beads can be observed as a reduction in the intensity in the flow through. (j) Stain-freeTM SDS–PAGE gel showing Col-0 extracts which were incubated with the HIS-HRV3C-GFP or TASH3_SH3 coupled beads. (k) Western blot detection using a general anti-Ubiquitin antibody (P4D1) showing Col-0 extracts which were incubated with the HIS-HRV3C-GFP or TASH3_SH3 coupled beads. The smear in the bound fraction indicates that the SH3 domain from TASH3 can bind ubiquitinated proteins as opposed to the HIS-HRV3C-GFP control. I – Input, FT - flow through, B – bound fractions.

Extended Data Fig. 8 Analysis of PM fraction purity.

(a) Immunoblot analysis of different fractions acquired using the Minute™ Plant Plasma Membrane Protein Isolation Kit. Collected fractions: total protein fraction (TP), nuclei and debris fraction (ND), cytosolic fraction (C), total membrane fraction (TM), organelle membrane fraction (OM) and plasma membrane fraction (PM). The obtained fractions were separated by SDS–PAGE, visualized using stain-free gel, blotted and probed with antibodies against the Aquaporin PIP2;7 (PIP2;7, a PM marker), cytosolic Ascorbate Peroxidase (cAPX, a cytosol marker) and Cytochrome C (CytC, a mitochondrial marker). Full-length bands of the proteins are marked with arrowheads (PIP2;7 – black arrowhead; cAPX – grey arrowhead; CytC – white arrowhead).The antibodies used are listed at the right bottom corner beneath the blots.

Source data

Supplementary information

Reporting Summary

Supplementary Data

Dataset 1: Quantitative analysis of GFP pulldown TPLATE-GFP (tplate) versus TPLATE-GFP (noshtplate) based on the MaxQuant proteingroups file of GFP pulldown samples analysed by LC–MS/MS on Q Exactive (Thermo Fisher Scientific). Dataset 2: Differential analysis of PM purified samples nosh versus Col-O, based on the MaxQuant proteingroups file of the PM samples analysed by LC–MS/MS on Q Exactive HF (Thermo Fisher Scientific). Dataset 3: Primers used for cloning and genotyping.

Supplementary Alignment File

Fasta file containing the sequences used to generate the phylogenetic tree in Extended Data Fig. 4c.

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Grones, P., De Meyer, A., Pleskot, R. et al. The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo. Nat. Plants 8, 1467–1483 (2022). https://doi.org/10.1038/s41477-022-01280-1

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