Targeted fluorescent probes in peroxisome function

Fluorescent peptides form a new generation of analytical tools for visualiz ing intracellular processes and molecular interactions at the level of sing le cells. The peptide-based reporters combine the sensitivity of fluorescen ce detection with the information specificity of amino acid sequences. Rece ntly we have succeeded in targeting a fluorescent heptapeptide (acetyl-CKGG AKL) carrying a peroxisomal targeting signal (PTS1) to peroxisomes in intac t 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 degreesC and typically visible in the cel l after 15 min or less. Cells lacking an active peroxisomal protein import system, as in the case of Zellweger syndrome, were stained diffusely throug hout the cell. Uptake of the peptide probes was not inhibited at 4 degreesC or when the cells were depleted of ATP. Under these conditions translocati on to peroxisomes was blocked. This indicates that the uptake by cells is d iffusion-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 memb rane was dissipated when an ionophore (CCCP) was added. In fibroblasts of c hondrodysplasia punctata patients with defects in the peroxisomal import of proteins carrying a PTS2 sequence, import of the PTS1-peptide probe into p eroxisomes appeared normal, but these peroxisomes have a pH of 6.8 equal to that of the cytosol. Coupling different fluorophores to the PTS1-peptide o ffers the possibility of determining in time and space as to how peroxisome s function in living cells.