Functional And Action Mechnisms Analysis Of Regulation Of AP-1-mediated Exocytosis By Adaptor Protein Complexes AP-2 And TPC In Arabidopsis

Clathrin-mediated endocytosis(CME)and exocytosis(CMX)are predominantpathways for the trafficking of membrane proteins in animal and plant cells.Plant clathrin co-localizes with its adaptor protein complexes,Adaptor Protein 2(AP-2)and the TPLATE complex(TPC)at the plasma membrane(PIM),and with AP-1 at the trans-Golgi network/early endosome(TGN/EE),which are essential for CME and CMX,respectively.AP-1 is a heterotetramer consisting of AP1γ,AP1/2β,AP1μ.,and AP1σ.Similar to AP-1,AP-2 as a heterotetrameric complex is composed of AP2α,AP1/2β,AP2μ,and AP2σ,whereas the TPC is a heterooctameric complex with TPLATE,TML,TASH3,LOLITA,TWD40-1,TWD40-2,AtEH1,and AtEH2.In animal cells,it is well known that endocytosis and exocytosis processes for plasma membrane proteins occur in a synergistic regulation manner.However,whether there exists a similar regulatory mechanism in plant cells is largely unknown.Our previous studies have shown that loss of clathrin light chain 2(CLC2)and CLC3 functions inhibited PM proteins endocytosis and recycling processes in Arabidopsis thaliana.In addition,plant hormone SA(salicylic acid)and synthetic CME inhibitor A23(TyrA23)regulate CME via modulating the PM recruitment of AP-2 subunits and clathrin,whereas auxin specifically regulates clathrin recruitment to the PM and thereby CME.In this study,laser scanning confocal microscopy,combined with genetic,cytological and pharmacological methods,was used to identify the effects of loss of AP-2 subunits or partial loss of TPC subunits on AP-1-mediated exocytosis pathway.The main results in this study were as follows:(1)Subcellular co-localization analysis showed that a high coefficient of co-localization was observed between clathrin and the TGN/EE markers,by contrast,an extremely low coefficient between clathrin and the Golgi or vacuole markers in plant cells,suggesting that intracellular clathrin predominantly localizes at the TGN/EE,which defines intracellular action site.(2)Genetic analysis revealed that loss of AP2μ or AP2σ function and partial loss of TPLATE or TML attenuated the recycling to the PM of PM proteins including PIN2-GFP and RCI2A-GFP and AP-1-mediated secretion/exocytosis of Sec-GFP,a secretion marker.(3)Pharmacological analysis showed that treatments with CME inhibitors including auxin(IAA and 2,4-D),SA,and TyrA23 enhanced intracellular accumulation of Sec-GFP,suggesting CME inhibitor impacts on the secretion/exocytosis of Sec-GFP.(4)Genetic and pharmacological analyses,revealed that loss of AP2μ or AP2σfunction,partial loss of TPLATE or TML,and CME inhibitor treatment impaired TGN/EE recruitment of AP-1 subunits AP1μ and AP1σ but did not affect subcellular localization of other TGN/EE markers.This study can conclude that clathrin predominantly functions at the PM and TGN/EE;AP-2 and TPC subunits regulate exocytosis/recycling/secretion processes of PM proteins via modulating TGN/EE recruitment of AP-1 subunits.These results demonstrate that AP-2 and TPC regulate AP-1-mediated exocytosis,and provide a new insight into CME regulation of CMX and membrane protein trafficking mechanisms in plant cells.