Multiple internal sorting determinants can contribute to the trafficking of cruciferin to protein storage vacuoles
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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.
KeywordsSeed storage protein Cruciferin Sorting determinants Protein storage vacuole
This work was supported with funding from the Agriculture and Agri-Food Canada Canadian Crop Genomics Initiative.
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- Bechtold N, Ellis J, Pelletier F (1993) In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci Paris Life Sci 316:1194–1199Google Scholar
- Ebine K, Okatani Y, Uemura T, Goh T, Shoda K, Niihama M, Morita MT, Spitzer C, Otegui MS, Nakano A, Ueda T (2008) A SNARE complex unique to seed plants is required for protein storage vacuole biogenesis and seed development of Arabidopsis thaliana. Plant Cell 20:3006–3021CrossRefPubMedCentralPubMedGoogle Scholar
- Inoue S (2006) Foundations of confocal scanned imaging in light microscopy. In: JB Pawley (ed) Handbook of biological confocal microscopy, 3rd Edn. Springer Science and Business Media, LLC.Google Scholar
- Matsuoka K, Neuhaus J-M (1999) Cis-elements of protein transport to the plant vacuoles. J Exp Biol 50:165–174Google Scholar
- Robinson DG, Bäumer M, Hinz G, Hohl I (1998) Vesicle transfer of storage proteins to the vacuole: the role of the Golgi apparatus and multivesicular bodies 152:659–667Google Scholar
- Shimada T, Yamada K, Kataoka M, Nakaune S, Koumoto Y, Kuroyanagi M, Tabata S, Kato T, Shinozaki K, Seki M, Kobayashi M, Kondo M, Nishimura M, Hara-Nishimura I (2003b) Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana. J Biol Chem 278:32292–32299CrossRefPubMedGoogle Scholar