Dataset Information

Molecular architecture of the endocytic TPLATE complex – part2 IP-MS dataset

ABSTRACT: Eukaryotic cells rely on endocytosis to regulate their plasma membrane proteome and lipidome. Most eukaryotic groups, except fungi and animals, have retained the evolutionary ancient TSET complex as endocytic regulator. Unlike other coatomer complexes, structural insight into TSET is lacking. Here, we reveal the molecular architecture of plant TSET (TPLATE complex/TPC) using an integrative structural approach. We identify crucial roles for specific TSET subunits in complex assembly and membrane interaction. Our data therefore generates novel evolutionary insight into the difference between the hexameric TSET in Dictyostelium and the octameric TPC in plants. Structural elucidation of this ancient adaptor complex represents the missing piece in the coatomer-puzzle and vastly advances our functional as well as evolutionary insight into the process of endocytosis.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Seedling

SUBMITTER: Dominique Eeckhout

LAB HEAD: Daniel Van Damme

PROVIDER: PXD023051 | Pride | 2021-03-04

REPOSITORIES: Pride

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Publications

Molecular architecture of the endocytic TPLATE complex.

Yperman Klaas K Wang Jie J Eeckhout Dominique D Winkler Joanna J Vu Lam Dai LD Vandorpe Michael M Grones Peter P Mylle Evelien E Kraus Michael M Merceron Romain R Nolf Jonah J Mor Eliana E De Bruyn Pieter P Loris Remy R Potocký Martin M Savvides Savvas N SN De Rybel Bert B De Jaeger Geert G Van Damme Daniël D Pleskot Roman R

Science advances 20210226 9

Eukaryotic cells rely on endocytosis to regulate their plasma membrane proteome and lipidome. Most eukaryotic groups, except fungi and animals, have retained the evolutionary ancient TSET complex as an endocytic regulator. Unlike other coatomer complexes, structural insight into TSET is lacking. Here, we reveal the molecular architecture of plant TSET [TPLATE complex (TPC)] using an integrative structural approach. We identify crucial roles for specific TSET subunits in complex assembly and memb ...[more]

PMID: 33637534

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Project description:Genomic rearrangements leading to intragenic gene fusion are mainly found in some types of haematopoietic malignancies and sarcomas. Recently they have been described also in carcinomas such as the papillary thyroid histotype (60%-70%) and the Hürthle thyroid tumours (58%). The presence of junction oncogene constitutes an area of exciting research for emerging therapy as targeting the RET-PTC1 fusion oncogene by using small interfering RNA (siRNA) strategies since it is present only in the tumour cells and not in the surrounding normal cells. Therefore, we developed a siRNA against RET-PTC1 junction and assess its efficiency on the human papillary thyroid carcinoma cell line TPC-1 which spontaneously harbours the RET-PTC1 oncogene. The targeted genes are assessed by microarray analysis by comparing the regulated genes by the siRNA_RET-PTC1 vs a siRNA_RET developed on the RET part of the mRNA minus the siRNA_control that contain four mutation within the RET-PTC1 sequence. To test the targeted genes in the TPC-1 cell line that spontaneously harbours RET-PTC1 junction of two siRNAs developed: Within the RET-PTC1 junction, and in the mRNA RET part. A non-specific siRNA harbouring 4 mutations within the RET-PTC1 sequence was used as negative control (siRNA_control). By real-time PCR (Q-RT-PCR) we demonstrated that both siRNAs (siRNA_RET-PTC1 and siRNA_RET) significantly reduce RET mRNA levels of about 85 % in TPC-1 cells. The negative control did not show an effect of RET mRNA levels. Three independent transfections were performed on TPC-1 cells using 5 µl of Lipofectamine 2000 transfection reagent (Invitrogen, Cergy-Pontoise, France) and 50nM of i) siRNA_RET-PTC1 or ii) siRNA_RET or iii) siRNA_control that harbour 4 mutations within its sequence. Total RNAs of untreated cells and transfected cells were purified using the RNA cleanup and concentration kit (QIAGEN, Hilden, Germany) and gathered in 4 pools : 1) TPC-1 harbouring RET-PTC1 ; 2) TPC-1 silenced for RET-PTC1 with siRNA RET-PTC1 ; 3) TPC-1 silenced for siRNA RET ; 4) TPC-1 treated with the siRNA control.

Dataset Information

Molecular architecture of the endocytic TPLATE complex – part2 IP-MS dataset

Publications

Molecular architecture of the endocytic TPLATE complex.

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