Signals of the Amaranth 11S globulin that are sufficient to target green fluorescent protein and beta-glucuronidase to the vacuoles

SILVANA PETRUCCELLI

Glasgow

Congreso; Symposium Meeting Membrane Trafficking in Plants. Organized by Society for Experimental Biology, University of Glasgow; 2003

SOCIETY FOR EXPERIMENTAL BIOLOGY

Signals of the Amaranth 11S globulin that are sufficient to target green fluorescent protein and beta-glucuronidase to the vacuoles. Storage proteins do not have any conserved motif that can function as vacuolar sorting determinants and it has been suggested that aggregation itself can function as a targeting signal.  The C-terminal tetrapeptide of phaseolin has been shown to be necessary and sufficient for targeting to vacuoles, but there are also indications, that this is not the only signal involved in the process and that the sorting machinery require cumulative information. The aim of this work was to identify signals involved in the vacuolar targeting of the Amaranth 11S protein.  Two different aminoacid sequences of amaranth 11S globulin were chosen. The first sequence is the C-terminal pentapeptide of amaranth 11S globulin, KISIA and the internal  sequence: GNIFRGF (Amh-H1N) that is similar to the sequence-specific vacuolar sorting determinant NPIR, responsible of directing proteins to lytic vacuoles.  This motif was named Helix 1 because corresponds to an alpha helix region found in the crystal structures of both 7S and 11S storage globulins. These signals were fused at the C terminal region of the genes encoding for green fluorescent protein (GFP) or GFP-GUS. To target these proteins to the secretory pathway a mouse immunoglobulin signal peptide was fused to their N-terminal sequence. These constructs were transient expressed in Arabidopsis protoplasts and were observed by confocal microscopy at different times after transfection. A typical vacuolar pattern was observed 36-48 hs after transfection, therefore both signals are sufficient to target both GFP and GFP-GUS to vacuoles and the targeting is independent of the size of the reporter gene.