Characterization of the trafficking of short PIN-FORMED Proteins

Abstract

PIN-FORMED (PIN) proteins are transmembrane proteins that have auxin efflux capabilities. Within Arabidopsis thaliana there exist eight PIN proteins, where PIN1-4, 6, and 7 (long PINs) contain long hydrophilic loop while PIN5 and 8 (short PINs) contain short hydrophilic loop. The trafficking of long PINs have been characterized where the internalization of long PINs via clathrin-mediated endocytosis is inhibited by auxin via AUXIN-BINDING PROTEIN1 (ABP1). However, the trafficking of short PINs have not yet been characterized. To determine the trafficking of short PINs, auxin and the inhibitor of clathrin, TyrA23, were treated to short PINs. Both natural auxin IAA and synthetic auxin NAA inhibited the short PINs localization to the plasma membrane. TyrA23 also inhibited the short PINs localization to the plasma membrane. The results indicate that instead of inhibiting the internalization of short PINs, auxin is inhibiting the localization of short PINs to the plasma membrane. To determine that auxin is not inhibiting the internalization of short PINs, the fungal toxin brefeldin A (BFA) was treated to PIN5 with auxin. When short PINs were treated with both auxin and BFA, auxin was not able to inhibit the formation of BFA bodies, indicating that the auxin is unable to inhibit the internalization of short PINs. To determine whether ABP1 is involved in short PINs trafficking, ABP1-specific auxin agonists, pC2-PAA, was treated to PIN5. pC2-PAA inhibited the PIN5 localization to the plasma membrane, indicating that ABP1 is able to dictate the localization of short PINs. The chemical treatments, along with the localization of ABP1 and clathrin on the trans-Golgi network (TGN), indicate that the secretion of short PINs is affected when treated with auxin. To determine whether the hydrophilic loop of the short PINs contains the necessary cues to inhibit short PINs PM localization under auxin treatment, the chimeric protein C1 (PIN5::PIN2-HL:GFP), which contains the hydrophilic loop of both PIN2 and PIN5, was treated with NAA. When treated with NAA, the C1 was unable to localize to the PM even in the existence of PIN2-HL suggesting that the molecular cue located at the PIN5-HL is able to mask the molecular cue of PIN2-HL