Trans-Golgi Network-Located AP1 Gamma Adaptins Mediate Dileucine Motif-Directed Vacuolar Targeting in Arabidopsis

Abstract

Membrane proteins on the tonoplast are indispensible for vacuolar functions in plants. However, how these proteins are transported to the vacuole and how they become separated from plasma membrane proteins remain largely unknown. In this study, we used Arabidopsis thaliana vacuolar ion transporter1 (VIT1) as a reporter to study the mechanisms of tonoplast targeting. We showed that VIT1 reached the tonoplast through a pathway involving the endoplasmic reticulum (ER), Golgi, trans-Golgi network (TGN), prevacuolar compartment, and tonoplast. VIT1 contains a putative N-terminal dihydrophobic type ER export signal, and its N terminus has a conserved dileucine motif (EKQTLL), which is responsible for tonoplast targeting. In vitro peptide binding assays with synthetic VIT1 N terminus identified adaptor protein complex-1 (AP1) subunits that interacted with the dileucine motif. A deficiency of AP1 gamma adaptins in Arabidopsis cells caused relocation of tonoplast proteins containing the dileucine motif, such as VIT1 and inositol transporter1, to the plasma membrane. The dileucine motif also effectively rerouted the plasma membrane protein SCAMP1 to the tonoplast. Together with subcellular localization studies showing that AP1 gamma adaptins localize to the TGN, we propose that the AP1 complex on the TGN mediates tonoplast targeting of membrane proteins with the dileucine motif.

The Plant Trans-Golgi Network: Not Just a Matter of Distinction

“…The Arabidopsis genome encodes putative subunits of all five AP complexes (Happel et al, 2004;Hirst et al, 2011;Park et al, 2013). Arabidopsis AP1 localizes to the TGN (Park et al, 2013;Wang et al, 2014), and mutants of the AP1M2 subunit are affected in the secretory and vacuolar transport pathways. In addition, trafficking of the essential SNARE KNOLLE from the TGN to the cell plate is impaired in dividing cells of ap1m2 mutants (Park et al, 2013).…”

Wheat Vacuolar Iron Transporter TaVIT2 Transports Fe and Mn and Is Effective for Biofortification

“…TaVIT1 also was found in the vacuolar membrane fraction, but based on higher abundance in the total fraction, it appeared that most of the TaVIT1 protein was targeted to other membranes. Closer inspection of the amino acid sequences revealed that TaVIT2 contains a universally conserved di-Leu motif for targeting to the vacuolar membrane (Bonifacino and Traub, 2003;Wang et al, 2014), which is absent from TaVIT1 (Supplemental Fig. S1B).…”

EXPO and Autophagosomes are Distinct Organelles in Plants

“…For each expressing construct, more than 20 individual cells were examined that represented .75% of the cells showing the same patterns or localizations. Pearson correlation coefficients were calculated using a Pearson and Spearman Correlation coefficients colocalization plug-in of the ImageJ program (National Institutes of Health; French et al, 2008;Wang et al, 2014;Gao et al, 2015). Images were processed via Adobe Photoshop as described previously (Jiang and Rogers, 1998).…”

“…For secondary antibodies, Alexa Fluor-568 anti-rabbit or anti-rat (Molecular Probes) was used at 1:1,000 dilutions for immunodetection. Pearson correlation coefficients were calculated from more than five individual cells by using a PSC colocalization plug-in of the ImageJ program (French et al, 2008;Wang et al, 2014;Gao et al, 2015), which produced r values in the range of 21 to +1, with 0 indicating no discernible correlation, and +1 and 21 indicating strong positive or negative correlations, respectively.…”