Site-directed mutations of the FAD-linked glycerophosphate dehydrogenase gene impairs the mitochondrial anchoring of the enzyme in transfected COS-7 cells.
COS-7 cells were transfected with the green fluorescent protein (GFP) of Aequorea victoria, human mitochondrial FAD-linked glycerophosphate dehydrogenase (mGDH), a mGDHwt-EGFP construct, or two mutant mGDH-proteins fused with EGFP. The site of mutation was selected to affect cationic amino acids in the peptide signal sequence currently believed to play a key role in the subcellular distribution of mitochondrial proteins. All proteins were suitably expressed in the COS-7 cells. However, an increase in mGDH enzymatic activity above the control value in non-transfected COS-7 cell homogenates was only observed in cells transfected with mGDH, indicating that the catalytic activity of mGDH was masked in fused proteins. Confocal microscopy documented that, in the cells transfected with the mGDHwt-EGFP construct, the fusion protein was located exclusively in mitochondria, this contrasting with the nuclear labelling of cells expressing the green fluorescent protein alone. The mitochondrial anchoring of the mutated mGDH fused protein was altered, this alteration being most obvious in the mGDH313233-EGFP mutant. These findings raise the idea that a conformation change of the mGDH protein, as resulting from either an inherited or acquired alteration of its amino acid sequence, may affect its subcellular distribution and, hence, modify its immunogenic potential.
Fabregat
M E
ME
Medical Department, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Hospital Clinic, Barcelona, Spain.
Usac
E F
EF
Franco
C
C
Enrich
C
C
Malaisse
W J
WJ
Gomis
R
R
Enric
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D005993:Glycerolphosphate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D037401:Scyphozoa; D014162:Transfection
D008164:Luminescent Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D009243:NAD; D049452:Green Fluorescent Proteins; D005993:Glycerolphosphate Dehydrogenase