Subcellular localization of oxidosqualene cyclases from Arabidopsis thaliana, Trypanosoma cruzi, and Pneumocystis carinii expressed in yeast.
Cycloartenol synthase from Arabidopsis thaliana and lanosterol synthase from Trypanosoma cruzi and Pneumocystis carinii were expressed in yeast, and their subcellular distribution in the expressing cells was compared. Determination of enzymatic (oxidosqualene cyclase, OSC) activity and SDS-PAGE analysis of subcellular fractions proved that enzymes from T. cruzi and A. thaliana have high affinity for lipid particles, a subcellular compartment rich in triacylglycerols, and steryl esters, harboring several enzymes of lipid metabolism. In lipid particles of strains expressing the P. carinii enzyme, neither OSC activity nor the electrophoretic band at the appropriate M.W. were detected. Microsomes from the three expressing strains retained some OSC activity. Affinity of enzymes from A. thaliana and T. cruzi for lipid particles is similar to that of OSC of Saccharomyces cerevisiae, which is mainly located in this compartment. A different distribution of OSC in yeast cells suggests that they differ in some structural features critical for the interaction with the surface of lipid particles. Computer analysis supports the hypothesis of the structural difference since OSC from S. cerevisiae, A. thaliana, and T. cruzi lack or contain only one transmembrane spanning domain (a structural feature that makes a protein poorly inclined to associate with lipid particles), whereas OSC from P. carinii possesses six transmembrane domains. In the strain expressing cycloartenol synthase from A. thaliana, the accumulation of lipid particles largely exceeded that of the other strains.
Milla
P
P
Università degli Studi di Torino, Dipartimento di Scienza e Tecnologia del Farmaco, 1-10125 Torino, Italy.
Viola
F
F
Oliaro Bosso
S
S
Rocco
F
F
Cattel
L
L
Joubert
B M
BM
LeClair
R J
RJ
Matsuda
S P T
SP
Balliano
G
G
D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism*; D045363:Pneumocystis carinii / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013347:Subcellular Fractions / Q000201:enzymology*; D014349:Trypanosoma cruzi / Q000201:enzymology*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D019751:Intramolecular Transferases; C021924:lanosterol synthase