Regulation of ABCB1/PGP1-catalysed auxin transport by linker phosphorylation

Abstract

Polar transport of the plant hormone auxin is controlled by PIN-and ABCB/PGP-efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co-immunoprecipitation (co-IP) and shotgun LC-MS/MS analysis, we identified PID as a valid partner in the interaction with TWD1. Invitro and yeast expression analyses indicated that PID specifically modulates ABCB1-mediated auxin efflux in an action that is dependent on its kinase activity and that is reverted by quercetin binding and thus inhibition of PID autophosphorylation. Triple ABCB1/PID/TWD1 co-transfection in tobacco revealed that PID enhances ABCB1-mediated auxin efflux but blocks ABCB1 in the presence of TWD1. Phospho-proteomic analyses identified S634 as a key residue of the regulatory ABCB1 linker and a very likely target of PID phosphorylation that determines both transporter drug binding and activity. In summary, we provide evidence that PID phosphorylation has a dual, counter-active impact on ABCB1 activity that is coordinated by TWD1-PID interaction.

The cyclophilin A DIAGEOTROPICA gene affects auxin transport in both root and shoot to control lateral root formation

“…To explore how DGT could functionally affect auxin transporters, we analyzed the colocalization of DGT with Arabidopsis PIN1 and ABCB1 upon co-expression in N. benthamiana leaves. When expressed alone, DGT localized predominantly in the nucleus, in addition to signals in the cytoplasm and the cell periphery as expected; PIN1 and ABCB1 localized predominantly to the plasma membrane, consistent with previous results (Henrichs et al, 2012) (Fig. 7D).…”

“…7B), indicating that DGT might act preferably as a negative regulator of PIN transporters. Because PIN1 is not functional in S. cerevisiae without ABCB1 (Blakeslee et al, 2007;Kim et al, 2010), we re-tested the effect of DGT on PIN1 in a tobacco (Nicotiana benthamiana) leaf transport system (Henrichs et al, 2012). Analogous to the yeast system, a mCherry-DGT fusion had a negative effect on PIN1-driven IAA efflux but no significant effect on ABCB1-driven IAA efflux, demonstrating a preferential regulation of PIN transporters (Fig.…”

Master and servant: Regulation of auxin transporters by FKBPs and cyclophilins

“…Functional FKBP-ABCB interaction seems to be conserved throughout evolution as judged from the fact that the activity of murine ABCB3/MDR3 transporter expressed in S. cerevisiae requires the presence of yeast FKBP12 [98]. Yeast FKBP12 also seems to compete with TWD1 for regulation of Arabidopsis ABCB1 activity in a S. cerevisiae auxin-transport system [43], which is further supported by the finding that FKBP12 partially complements the twd1 mutant phenotype when expressed in Arabidopsis [99].…”

Inactivation of Plasma Membrane–Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response inArabidopsis

“…By contrast, the pgp4 mutation impairs both acro-and basipetal auxin transport only in lateral roots without affecting the elongation and gravitropic response of primary root . Despite these apparently contradictory observations, based on the finding that some ABCB/PGPs, such as PGP1 (Henrichs et al, 2012), are phosphorylated by the AGC PIN kinase PID, it will be interesting to determine whether CRK5 or other members of the Arabidopsis CRK family contribute by phosphorylation to the modulation of regulatory interactions between ABCB/PGPs and PINs (Bandyopadhyay et al, 2007;Titapiwatanakun and Murphy, 2009). Definition of the role of CRK5 in the regulation of root gravitropic response now opens the way to answer these important questions by combined genetic analysis of members of the CRK family.…”

“…Emerging data indicate that cortical actin accumulation regulates clathrin-dependent endocytosis Nagawa et al, 2012), whereas enhanced inositol triphosphate and Ca 2+ levels decelerate exocytosis of PIN1 and PIN2 similarly to mutations of inositol polyphosphate 1-phosphatase and phosphatidylinositol monophosphate 5-kinase genes (Zhang et al, 2011;Mei et al, 2012). Furthermore, signaling through the 3-phosphoinositidedependent kinase1 and interactions with Ca 2+ binding or calmodulinlike proteins appear to regulate the activity of AGC kinases that phosphorylate central hydrophilic loops of PINs, as well as ABCB/ PGPs (Benjamins et al, 2003;Zegzouti et al, 2006;Henrichs et al, 2012;Rademacher and Offringa, 2012).…”