Targeted fluorescent probes in peroxisome function.
Fluorescent peptides form a new generation of analytical tools for visualizing intracellular processes and molecular interactions at the level of single cells. The peptide-based reporters combine the sensitivity of fluorescence detection with the information specificity of amino acid sequences. Recently we have succeeded in targeting a fluorescent heptapeptide (acetyl-CKGGAKL) carrying a peroxisomal targeting signal (PTS1) to peroxisomes in intact cells. The fluorophores conjugated to the PTS1-peptide were fluorescein, BODIPY and the pH-sensitive SNAFL-2. When added to cells, these fluorescent peptides were internalized at 37 degrees C and typically visible in the cell after 15 min or less. Cells lacking an active peroxisomal protein import system, as in the case of Zellweger syndrome, were stained diffusely throughout the cell. Uptake of the peptide probes was not inhibited at 4 degrees C or when the cells were depleted of ATP. Under these conditions translocation to peroxisomes was blocked. This indicates that the uptake by cells is diffusion-driven and not an active process. Using the SNAFL-2-PTS1 peptide, we established by ratio-imaging that peroxisomes of human fibroblasts have an internal pH of 8.2. The concurrent pH gradient over the peroxisomal membrane was dissipated when an ionophore (CCCP) was added. In fibroblasts of chondrodysplasia punctata patients with defects in the peroxisomal import of proteins carrying a PTS2 sequence, import of the PTS1-peptide probe into peroxisomes appeared normal, but these peroxisomes have a pH of 6.8 equal to that of the cytosol. Coupling different fluorophores to the PTS1-peptide offers the possibility of determining in time and space as to how peroxisomes function in living cells.
Dansen
T B
TB
Institute of Biomembranes, Centre for Biomembranes and Lipid Enzymology, Department of Biochemistry of Lipids, Utrecht University, The Netherlands.
Pap EHW
Wanders
R J
RJ
Wirtz
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D001896:Boron Compounds; D002806:Chondrodysplasia Punctata / Q000378:metabolism; D015195:Drug Design; D005347:Fibroblasts / Q000378:metabolism; D019793:Fluorescein; D005452:Fluoresceins; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015336:Molecular Probe Techniques; D009842:Oligopeptides / Q000737:chemistry; D020675:Peroxisomes / Q000378:metabolism*; D021382:Protein Sorting Signals
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009842:Oligopeptides; D021382:Protein Sorting Signals; C401959:seminaphthofluorescein; D019793:Fluorescein