Identification and characterization of a eukaryotically encoded rubredoxin in a cryptomonad alga.
We have identified an open reading frame with homology to prokaryotic rubredoxins (rds) on a nucleomorph chromosome of the cryptomonad alga Guillardia theta. cDNA analysis let us propose that the rd preprotein has an NH(2)-terminal extension that functions as a transit peptide for import into the plastid. Compared to rds found in non-photosynthetic prokaryotes or found in bacteria that exhibit an anoxigenic photosynthesis apparatus, nucleomorph rd has a COOH-terminal extension, which shows high homology exclusively to the COOH-termini of cyanobacterial rds as well as to a hypothetical rd in the Arabidopsis genome. This extension can be divided into a putative membrane anchor and a stretch of about 20 amino acids with unknown function linking the common rd fold to this anchor. Overexpression of nucleomorph rd in Escherichia coli using a T7 RNA polymerase/promotor system resulted in a mixture of iron-containing holorubredoxin and zinc-substituted protein. Preliminary spectroscopic studies of the iron form of nucleomorph rd suggest the existence of a native rd-type iron site. One-dimensional nuclear magnetic resonance spectroscopy of recombinant Zn-rd suggests the presence of a stable tertiary fold similar to that of other rd structures determined previously.
Wastl
J
J
Cell Biology and Applied Botany, Philipps-University Marburg, Karl-von-Frisch-Strasse, D-35032, Marburg, Germany.
Sticht
H
H
Maier
U G
UG
Rösch
P
P
Hoffmann
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D002467:Cell Nucleus / Q000235:genetics; D003001:Cloning, Molecular; D056890:Eukaryota / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism; D005057:Eukaryotic Cells / Q000166:cytology*; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D015388:Organelles / Q000235:genetics*; D018087:Plastids / Q000378:metabolism; D011387:Prokaryotic Cells / Q000737:chemistry; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012416:Rubredoxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013559:Symbiosis / Q000235:genetics; D015032:Zinc / Q000378:metabolism
D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012416:Rubredoxins; D007501:Iron; D015032:Zinc