The role of clathrin in exocytosis and the mutual regulation of endo- and exocytosis in plant cells

Abstract

Within the plant endomembrane system, the vesicle coat protein clathrin localizes to the plasma membrane (PM) and the trans-Golgi Network/Early Endosome (TGN/EE). While the role of clathrin as a major component of endocytosis at the PM is well established, its function at TGN/EE, possibly in exocytosis or the vacuolar pathway, is a matter of debate. This shared function of clathrin also opens a question whether plant cells possess a homeostatic mechanisms that balance rates of opposite trafficking routes, such as endo- and exocytosis. Here we address these questions using lines inducibly silencing CLATHRIN HEAVY CHAIN (CHC). We find a relocation of exocytic soluble and integral membrane protein cargoes to the vacuole, supporting a function of clathrin in exocytosis. A comparison with lines overexpressing AUXILIN-LIKE1, where inhibition of CME precedes rerouting of secretory cargoes, does not support a homeostatic regulatory mechanism adjusting exocytosis to the rates of endocytosis. Complementary experiments reveal only minor and variably detectable reductions in the rates of CME in secretory mutants, also not indicative of a converse homeostatic mechanism adjusting rates of endocytosis to the rates of secretion.

Developmental patterning function of GNOM ARF-GEF mediated from the cell periphery

“…Overall, in the course of experiments presented in this study, the localization of GN to the PM was most reliably observed with TIRF microscopy, rather than by CLSM; as discussed above, somewhat analogically, CCPs are always observed at the PM with TIRF microscopy, but the detection of fluorescent protein fusions of CLC at the PMs of RAM epidermis by CLSM is variable (e.g. Adamowski et al, 2018 , Adamowski et al, 2021b ).…”

“…We note that the detection of CLC markers by CLSM at the PMs of RAM epidermis is similarly limited (e.g. Adamowski et al, 2018 , Adamowski et al, 2021b ) despite CME being a housekeeping process.…”

“…As an independent verification, we introduced into GN pro :GN-GFP VAN3 pro :VAN3-mRFP an estradiol-inducible artificial microRNA construct down-regulating the expression of CLATHRIN HEAVY CHAIN (CHC ) genes, which encode a key structural component of the clathrin coats. Following silencing of CHC, secretion is inhibited and secretory cargoes are re-routed to the vacuole ( Adamowski et al, 2021b ). In XVE»amiCHCa , the BFA-induced relocation of GN-GFP to the PM too, was inhibited ( Figure 5B and F , and Figure 5—figure supplement 1B ).…”

Mapping the adaptor protein complex interaction network in Arabidopsis identifies P34 as a common stability regulator

“…Therefore, P34 might be a regulator of multiple clathrin-mediated vesicle trafficking pathways in Arabidopsis. Although debatable, some evidence exists that the activities of clathrin-dependent post-Golgi trafficking and CME are highly coordinated in plant cells 50, 63 . Recently, the recruitment of several endocytic components to the PM, including CLC1, CHC1, AP2M, AP2S, TPLATE and TML, was shown to be reduced in the ap1m2 mutant, but their protein levels to remain unchanged 50 .…”

Recent advances in plant endomembrane research and new microscopical techniques

“…Different views co-exist in the literatures concerning what is (or are) the secretory domain(s) of TGN. For example, the clathrin domain was defined as the secretory domain (Adamowski et al, 2021;Shimizu et al, 2021), while other studies suggested uncoated vesicles as part of the secretion process. The TGN protein ECHIDNA is sitting on uncoated vesicles (Boutte et al, 2013) and is clearly involved in secretion of auxin carriers and cell wall components while playing no role in endocytosis or endocytic recycling (Gendre et al, 2011(Gendre et al, , 2013.…”