Golgi-mediated vacuolar sorting in plant cells: RMR proteins are sorting receptors for the protein aggregation/membrane internalization pathway

The mechanism by which storage proteins carrying C-terminal vacuolar sorting determinants (ctVSDs) are sorted to vacuoles in plant cells has been controversial, and some reports suggest that receptors of the BP80/AtVSR family are responsible. Here we show that an Arabidopsis receptor homology-transmembrane-RING H2 (RMR) protein binds specifically the tobacco chitinase ctVSD both in vitro and in vivo, but not the proaleurain sequence-specific vacuolar sorting determinant (ssVSD). In contrast, BP80 binds the proaleurain ssVSD but not the chitinase ctVSD in vivo. In co-expression studies with red- and green-tagged forms of reporter proteins carrying the RMR or BP80 cytoplasmic tails, RMR protein and BP80 do not co-localize, as compared to BP80–AtVSR1 or RMR–RMR co-expressions. RMR protein co-localizes with the green fluorescent protein–chitinase ctVSD reporter while AtVSR1 does not. In transient expression experiments, reporters with the RMR protein cytoplasmic tail are internalized into vacuoles while those carrying the BP80 or AtVSR1 cytoplasmic tails are not. We propose that aggregation of soluble proteins carrying a ctVSD is accompanied by RMR binding and membrane internalization. Binding of aggregates would explain how the RMR protein, which does not recycle back to the Golgi, could serve as an efficient sorting receptor.