Correct targeting of a vacuolar tobacco chitinase in Saccharomyces cerevisiae--post-translational modifications are dependent on the host strain.
The chitinase gene FB7-1 of Nicotiana tabacum cv. samsun line 5 was expressed in the two Saccharomyces cerevisiae strains, INVSC2 and H4, under the control of the GAL1 promoter from S. cerevisiae and a multicopy plasmid vector. Both yeast strains express the plant gene as enzymatic active proteins. In transformants of the strain INVSC2, 94% of the total plant chitinase is contained inside the cells, probably within the vacuole which has been confirmed by subcellular fractionation as well as immunohistochemical experiments. This retention inside the cells is due to the C-terminally located 7 amino acids long vacuolar targeting peptide of the prochitinase. When this sequence was removed, chitinase was transported into the culture medium. Pulse-chase experiments revealed that during translation in transformants of both yeast strains one chitinase polypeptide can be immunoadsorbed with specific antibodies. In the case of INVSC2-transformants, newly formed chitinase is modified in a 60 min chase to slightly increase its molecular mass, whereas in H4-transformants the molecular mass constantly remained 32 kDa. By Western blot analysis two chitinase corresponding polypeptides of 32 and 37 kDa were accumulated in the culture medium of both transformants carrying the chitinase gene without the vacuolar targeting sequence. The larger one was very likely O-glycosylated. Whereas, both polypepitdes were also detected in cell extracts of the H4-transformant, only the smaller one was found in the INVSC2-transformant. The plant chitinase passed through the endoplasmic reticulum on its way to the vacuole. The N-terminal signal peptide responsible for the uptake into the endoplasmic reticulum is cleaved correctly. However, cleavage of the vacuolar targeting peptide located at the C-terminus, to give the mature chitinase is obviously influenced by the genetic background of the host strain. In INVSC2-transformants chitinase accumulates in its mature form whereas both the polypeptides of H4-transformants retain their vacuolar targeting peptide. Our results demonstrate that in the case of plant class I chitinase, the plant sorting signal is recognized in yeast cells but post-translational modifications are influenced by the host strain.
Kunze
I
I
Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany. kunzei@ipk-gatersleben.de
Nilsson
C
C
Adler
K
K
Manteuffel
R
R
Horstmann
C
C
Bröker
M
M
Kunze
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002688:Chitinases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli; D007150:Immunohistochemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000648:ultrastructure; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics; D014617:Vacuoles / Q000201:enzymology* / Q000648:ultrastructure
D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002688:Chitinases