Cellular localisation by immunolabelling and transmission electron microscopy of oxaloacetate decarboxylase or its individual subunits synthesised in Escherichia coli.
The genes oadGAB encoding the oxaloacetate decarboxylase gamma, alpha and beta-subunits from Klebsiella pneumoniae were expressed in Escherichia coli. Using different expression vectors, the entire enzyme or its individual subunits were synthesised. The expression was evidenced immunologically in whole cells with polyclonal antibodies raised against the purified oxaloacetate decarboxylase. The expressed alpha-subunit or a combination of alpha and beta-subunits were shown to reside in the cytoplasm, while the entire oxaloacetate decarboxylase or a gammaalpha-complex were located mostly in the cytoplasmic membrane. Interestingly, overexpression of the gammaalpha-complex or the entire oxaloacetate decarboxylase in E. coli led to a significant immunogold labelling in the cytoplasm, indicating that the alpha-subunit was not completely complexed to the membrane-bound gamma or betagamma-subunits.
Di Berardino
M
M
Mikrobiologisches Institut, Eidgenossische Technische Hochschule, Zurich, Switzerland.
Hermann
R
R
Dimroth
P
P
D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007150:Immunohistochemistry / Q000379:methods*; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics; D016253:Microscopy, Immunoelectron / Q000379:methods*; D010957:Plasmids; D011487:Protein Conformation
D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase