title: Multiple internal sorting determinants can contribute to the trafficking of cruciferin to protein storage vacuoles.
abstract: Trafficking of seed storage proteins to protein storage vacuoles is mediated by carboxy terminal and internal sorting determinants (ISDs). Protein modelling was used to identify candidate ISDs residing near surface-exposed regions in Arabidopsis thaliana cruciferin A (AtCruA). These were verified by AtCruA fusion to yellow fluorescent protein (YFP) and expression in developing embryos of A. thaliana. As the presence of endogenous cruciferin was found to mask the effects of weaker ISDs, experiments were conducted in a line that was devoid of cruciferin. In total, nine ISDs were discovered and a core determinant defined using a series of alanine scanning and deletion mutant variants. Coupling of functional data from AtCruA ISD-YFP fusions with statistical analysis of the physiochemical properties of analogous regions from several 11/12S globulins revealed that cruciferin ISDs likely adhere to the following rules: (1) ISDs are adjacent to or within hydrophilic, surface-exposed regions that serve to present them on the protein's surface; (2) ISDs generally have a hydrophobic character; (3) ISDs tend to have Leu or Ile residues at their core; (4) ISDs are approximately eight amino acids long with the physiochemical consensus [hydrophobic][preferably charged][small or hydrophobic, but not tiny][IL][polar, preferably charged][small, but not charged][hydrophobic, not charged, preferably not polar][hydrophobic, not tiny, preferably not polar]. Microscopic evidence is also presented for the presence of an interconnected protein storage vacuolar network in embryo cells, rather than discreet, individual vacuoles.
authors.affiliation: Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, S7N 0X2, Canada, Dwayne.Hegedus@agr.gc.ca.
authors.forename: Dwayne D
authors.initials: DD
authors.lastname: Hegedus
authors.forename: Cathy
authors.initials: C
authors.lastname: Coutu
authors.forename: Myrtle
authors.initials: M
authors.lastname: Harrington
authors.forename: Brad
authors.initials: B
authors.lastname: Hope
authors.forename: Kelsey
authors.initials: K
authors.lastname: Gerbrandt
authors.forename: Ivo
authors.initials: I
authors.lastname: Nikolov
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D004252:DNA Mutational Analysis; D021621:Imaging, Three-Dimensional; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D030821:Plants, Genetically Modified; D021381:Protein Transport; D055314:Seed Storage Proteins / Q000378:metabolism*; D012639:Seeds / Q000166:cytology / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D055314:Seed Storage Proteins; C571789:cruciferin A, Arabidopsis; C054562:yellow fluorescent protein, Bacteria