Fluorescence analysis of the Hansenula polymorpha peroxisomal targeting signal-1 receptor, Pex5p.
Correct sorting of newly synthesized peroxisomal matrix proteins is dependent on a peroxisomal targeting signal (PTS). So far two PTSs are known. PTS1 consists of a tripeptide that is located at the extreme C terminus of matrix proteins and is specifically recognized by the PTS1-receptor Pex5p. We studied Hansenula polymorpha Pex5p (HpPex5p) using fluorescence spectroscopy. The intensity of Trp fluorescence of purified HpPex5p increased by 25% upon shifting the pH from pH 6.0 to pH 7.2. Together with the results of fluorescence quenching by acrylamide, these data suggest that the conformation of HpPex5p differs at these two pH values. Fluorescence anisotropy decay measurements revealed that the pH affected the oligomeric state of HpPex5p, possibly from monomers/dimers at pH 6.0 to larger oligomeric forms at pH 7.2. Addition of dansylated peptides containing a PTS1, caused some shortening of the average fluorescence lifetime of the Trp residues, which was most pronounced at pH 7.2. Our data are discussed in relation to a molecular model of HpPex5p based on the three-dimensional structure of human Pex5p.
Boteva
Raina
R
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Koek
Anne
A
Visser
Nina V
NV
Visser
Antonie J W G
AJ
Krieger
Elmar
E
Zlateva
Theodora
T
Veenhuis
Marten
M
van der Klei
Ida
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D008958:Models, Molecular; D000074435:Peroxisome-Targeting Signal 1 Receptor; D010843:Pichia / Q000378:metabolism*; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins