Regulation Mechanisms Of Clathrin And Its Adaptor Proteins AP2 And TPC Recruitment To The Plasma Membrane In ARABIDOPSIS

Clathrin-mediated endocytosis(CME)is a predominant endocytic pathway for membrane protein internalization from the plasma membrane(PM)in eukaryotic cells.In plants,CME is essential for many developmental processes,nutrient uptake,polar auxin transport,and stress responses.Clathrin recruitment to the PM is dependent on its adaptor protein complexes AP2 and the TPC(TPLATE complex).AP2 is a heterooligomeric adaptor complex with ?,?,?,and ? whereas the TPC is an eight-core-component complex consisting of TPLATE,TML,TASH3,LOLITA,TWD40-1,TWD40-2,AtEH1,and AtEH2.Previous studies have shown that the plant hormones auxin,salicylic acid(SA)and CME inhibitor TyrA23 can rapidly inhibit the CME of many PM proteins,including the PIIN-FORMED(PIN)auxin efflux transporters and the water channel protein PLASMA MEMBRANE INTRINSIC PROTEIN2A(PIP2A).However,whether these CME inhibitors rapidly inhibit the CME of PM proteins via modulating the PM recruitment of clathrin,AP2 and the TPC remains to be defined.Our previous work has shown that exogenous auxin treatment differentially regulates CHC and CLC recruitment to the PM,indicating that auxin controls internalization of PM proteins via regulating clathrin recruitment to the PM.In this study,immunofluorescence microscopy and live-cell imaging,combined with genetic,cytological and pharmacological methods,were used to dissect mechanisms of the effects of auxin,SA,and TyrA23 on the recruitment to the PM of clathrin,AP2,and the TPC.The main results in this study are as follows:(1)Similar to the auxin effects,TyrA23(A23;30 ?M)and SA(25 ?M)regulate recruitment to the PM of CHC and CLC differentially.The results showed that A23 rapidly inhibited CLC recruitment to the PM but enhanced CHC recruitment upon 5-30 min treatment;in contrast,while CLC PM association was subsequently reverted back to the control level and the CHC level decreased 120 min after A23 exposure.SA short-term treatment had similar effects to auxin and A23 treatments.However,in SA long-term treatment,until after 240 min,the restoration of CLC PM association and inhibition of CHC PM association were observed.These results at least suggest that A23 and SA regulate CHC recruitment and thereby CME via rapidly modulating CLC recruitment to the PM.(2)Short-term treatments(30 min)with SA or A23 affected the membrane recruitment of AP1/2?1 and AP2? but not that of AP2? and AP2?.In long-term(120 min)treatment,A23 inhibited the recruitment to the PM of all of AP2 four subunits,whereas no differences were observed in AP2 recruitment to the PM between in SA long-term(120 min)and short-term(30 min)treatments.In addition,auxin(2,4-D;10 ?M)did not affect the membrane recruitment of all AP2 subunits in long-and short-term treatments.(3)The membrane association of the TPC subunits was not altered after auxin,SA,and Tyr A23 treatments(30 min),suggesting that these three CME inhibitors regulate clathrin membrane recruitment in an TPC-independent manner.However,in the presence of A23,impairment of TPC inhibited the recovery of CLC recruitment,indicating that the TPC is necessary for clathrin membrane recruitment in AP2-deficient cells.(4)Loss of AP2? or AP2? significantly inhibited the endocytosis of PIN2-GFP.However,SA and A23 failed to affect the endocytosis of PIN2-GFP in ap2? mutants;by contrast,SA and A23 effectively blocked PIN2-GFP endocytosis.In addition,auxin inhibited PIN2-GFP endocytosis in ap2? and ap2? mutants,indicating that AP2? may act as an important action target site for SA and A23 to regulate AP2 function.Based on these data,a preliminary conclusion was obtained as following:SA and A23 may regulate CME via modulating the PM recruitment of AP2 and clathrin,whereas auxin specially regulates clathrin and thereby CME.These data will provide new insights into molecular mechanisms of CME and is also important for studying the endocytosis of important PM proteins.