The plant vacuolar protein, phytohemagglutinin, is transported to the vacuole of transgenic yeast.
Phytohemagglutinin (PHA), the major seed lectin of the common bean, Phaseolus vulgaris, accumulates in the parenchyma cells of the cotyledons. It has been previously shown that PHA is cotranslationally inserted into the endoplasmic reticulum with cleavage of the NH2-terminal signal peptide. Two N-linked oligosaccharide side chains are added, one of which is modified to a complex type in the Golgi apparatus. PHA is then deposited in membrane-bound protein storage vacuoles which are biochemically and functionally equivalent to the vacuoles of yeast cells and the lysosomes of animal cells. We wished to determine whether yeast cells would recognize the vacuolar sorting determinant of PHA and target the protein to the yeast vacuole. We have expressed the gene for leukoagglutinating PHA (PHA-L) in yeast under control of the yeast acid phosphatase (PHO5) promoter. Under control of this promoter, PHA-L accumulates to 0.1% of the total yeast protein. PHA-L produced in yeast is glycosylated as expected for a yeast vacuolar glycoprotein. Cell fractionation studies show that PHA-L is efficiently transported to the yeast vacuole. This is the first demonstration that vacuolar targeting information is recognized between two highly divergent species. A small proportion of yeast PHA-L is secreted which may be due to inefficient recognition of the vacuolar sorting signal because of the presence of an uncleaved signal peptide on a subset of the PHA-L polypeptides. This system can now be used to identify the vacuolar sorting determinant of a plant vacuolar protein.
Tague
B W
BW
Department of Biology, University of California, San Diego, La Jolla 92093.
Chrispeels
M J
MJ
D001692:Biological Transport; D005822:Genetic Vectors; D009940:Organoids / Q000378:metabolism*; D010835:Phytohemagglutinins / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013104:Spheroplasts / Q000378:metabolism; D014170:Transformation, Genetic; D014617:Vacuoles / Q000378:metabolism*
D010835:Phytohemagglutinins