Degradation of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in the vacuole.
To understand how plant cells exert quality control over the proteins that pass through the secretory system we examined the transport and accumulation of the bean (Phaseolus vulgaris L.) vacuolar storage protein phaseolin, structurally modified to contain a helix-breaking epitope and carboxyterminal HDEL, an endoplasmic reticulum (ER)-retention signal. The constructs were expressed in tobacco (Nicotiana tabacum L.) with a seed-specific promoter. The results show that phaseolin-HDEL accumulates in the protein-storage vacuoles, indicating that HEDL does not contain sufficient information for retention in the ER. However, the ER of seeds expressing the phaseolin-HDEL construct contain relatively more phaseolin-HDEL compared to phaseolin in the ER of seeds expressing the phaseolin construct. This result indicates that the flow out of the ER is retarded but not arrested by the presence of HDEL. Introduction into phaseolin of the epitope "himet" (Hoffman et al., 1988, Plant Mol. Biol. 11, 717-729) greatly reduces the accumulation of HiMet phaseolin compared to normal phaseolin. However, the increased abundance within the ER is similar for both phaseolin-HDEL and HiMet phaseolin-HDEL. Using immunocytochemistry with specific antibodies, HiMet phaseolin was found in the ER, the Golgi stack, and in transport vesicles indicating that it was transport competent. It was also present at an early stage of seed development in the protein-storage vacuoles, but was not found there at later stages of seed development. Together these results support the conclusion that the HiMet epitope did not alter the structure of the protein sufficiently to make it transport incompetent.
Pueyo
J J
JJ
Department of Biology, University of California, San Diego, La Jolla 92093-0116, USA.
Chrispeels
M J
MJ
Herman
E M
EM
D000595:Amino Acid Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism*; D010947:Plants, Toxic; D021382:Protein Sorting Signals / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000378:metabolism; D014026:Nicotiana; D014170:Transformation, Genetic; D014617:Vacuoles / Q000378:metabolism*
D010940:Plant Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C048826:phaseolin protein, Phaseolus vulgaris