Conditional destabilization of the TPLATE complex impairs endocytic internalization

Wang, Jie; Yperman, Klaas; Grones, Peter; Jiang, Qihang; Dragwidge, Jonathan Michael; Mylle, Evelien; Mor, Eliana; Nolf, Jonah; Eeckhout, Dominique; Jaeger, Geert De; Rybel, Bert De; Pleskot, Roman; Damme, Daniël Van

doi:10.1073/pnas.2023456118

Cited by 24 publications

(50 citation statements)

References 67 publications

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“…To test this notion, we looked directly at CCVs in plant cells subjected to TPC disruption. For these studies, we used the inducible TPLATE loss-offunction mutant WDXM2 in which tplate mutant plants are complemented with a genetically destabilized version of TPLATE that after heat shock results in the total aggregation of TPLATE away from the PM and blocks CME (23). First, we further confirmed that heat shock treatment had no significant effect on the efficiency and kinetics of CME in plants by examining FM4-64 uptake and the dynamics of single CME events (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 82%

“…Thus, to test if the TPC itself could be involved in membrane remodeling, we purified the EH domains of the TPC member AtEH1/Pan1 and showed that they have intrinsic membrane-remodeling activity in vitro, therefore demonstrating that the TPC possess membranebending machinery. However, how this activity could be mediated in vivo remains an open question, as AtEH1/Pan1 remains associated with the PM following TPC disruption (23) but is not sufficient to generate the invagination (Fig. 3).…”

Section: Discussionmentioning

confidence: 99%

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The TPLATE complex mediates membrane bending during plant clathrin–mediated endocytosis

Johnson

Dahhan

Gnyliukh

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin–mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells.

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Section: Resultsmentioning

confidence: 82%

Section: Discussionmentioning

confidence: 99%

The TPLATE complex mediates membrane bending during plant clathrin–mediated endocytosis

Johnson

Dahhan

Gnyliukh

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…To test this notion, we looked directly at CCVs in plant cells subjected to TPC disruption. For these studies, we used the inducible TPLATE loss-of-function mutant WDXM2; where tplate mutant plants are complemented with a genetically destabilized version of TPLATE that after heat shock results in the total aggregation of TPLATE away from the PM and blocks CME (20). First, we further confirmed that the heat shock treatment had no significant effect on the efficiency and kinetics of CME in plants by examining FM4-64 uptake and the dynamics of single CME events (Fig.…”

mentioning

confidence: 73%

“…This demonstrated that AtEH1/Pan1 has the capacity to contribute to membrane bending. However, as AtEH1/Pan1 remains associated with the PM during WDXM2 disruption (20), but is not sufficient to generate the invagination (Fig. 3), it suggests that additional factors are required to modulate the membrane bending activity of the AtEH1/Pan1 EH domains.…”

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confidence: 99%

The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis

Johnson

Dahhan

Gnyliukh

et al. 2021

Preprint

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Clathrin-mediated endocytosis in plants is an essential process but the underlying mechanisms are poorly understood, not least because of the extreme intracellular turgor pressure acting against the formation of endocytic vesicles. In contrast to other models, plant endocytosis is independent of actin, indicating a mechanistically distinct solution. Here, by using biochemical and advanced microscopy approaches, we show that the plant-specific TPLATE complex acts outside of endocytic vesicles as a mediator of membrane bending. Cells with disrupted TPLATE fail to generate spherical vesicles, and in vitro biophysical assays identified protein domains with membrane bending capability. These results redefine the role of the TPLATE complex as a key component of the evolutionarily distinct mechanism mediating membrane bending against high turgor pressure to drive endocytosis in plant cells.One Sentence SummaryWhile plant CME is actin independent, we identify that the evolutionarily ancient octameric TPLATE complex mediates membrane bending against high turgor pressure in plant clathrin-mediated endocytosis.

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“…Plants have shown evolutionary divergence in their key EAPs compared to other model systems including, for example, the TPLATE complex (TPC), which likely evolved in early eukaryotes but is notably absent from yeast and metazoans and which is essential for plant clathrin-mediated endocytosis (Gadeyne et al, 2014;Hirst, et al, 2014;Wang, et al, 2021;Bashline, et al, 2015). Similar to AP-2, TPC functions exclusively at the PM and acts as a central interaction hub required for the formation of endocytic CCVs (Gadeyne, et al, 2014;Wang, et al 2016;Zhang, et al, 2015;Yperman, et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components

Dahhan¹,

Reynolds²,

Cárdenas³

et al. 2021

Preprint

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In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

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Conditional destabilization of the TPLATE complex impairs endocytic internalization

Cited by 24 publications

References 67 publications

The TPLATE complex mediates membrane bending during plant clathrin–mediated endocytosis

The TPLATE complex mediates membrane bending during plant clathrin–mediated endocytosis

The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis

Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components

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