Figure 1. Peroxisomal protein import using purified peroxisomes. Cells are homogenized to break the plasma membrane but not the organelle membranes. The homogenate, before or after removal of the cell debris and unbroken cells by low-speed centrifugation, is loaded onto a density gradient (Nycodenz or sucrose) and centrifuged at high speed up to 100 000g to separate peroxisomes (yellow) from other cell components. The fractions containing peroxisomes are collected and the peroxisomes are concentrated by centrifugation. The purified peroxisomes are incubated for 30 min at 25°C with the in vitro-translated radiolabelled peroxisomal protein of interest (shown in red). After incubation, the peroxisomes are collected by centrifugation and analysed by SDS-PAGE and autoradiography. Import of the radiolabelled proteins is determined first by measuring the radioactivity that spins down with the peroxisomal pellet, second by measuring resistance to proteases (imported proteins are protected by the peroxisomal membrane), and third by decrease in molecular weight (only in the case of some PTS2 proteins that are cleaved following import). To check that the protein does not fold into a compact protease-resistant form without being imported, the peroxisomal membrane can be broken with detergent (e.g. Triton X-100) before adding protease.

Figure 2. Peroxisomal protein import using living cells. cDNAs encoding normal or mutant proteins are cloned into appropriate plasmids to allow expression in yeast or mammalian cells. Yeast cells are usually stably transformed, whereas mammalian cells are usually transiently transfected, following a variety of possible techniques, such as electroporation or intranuclear microinjection. After culturing the cells for a few days, the cells are fixed, permeabilized, and immunostained using antibodies against the protein of interest and against a peroxisomal marker protein, such as catalase. On occasions an autofluorescent reporter (e.g. green fluorescent protein, GFP) can be used. Secondary immunostaining using fluorochromes of different colours (e.g. fluorescein, green; Texas red, red) enables the subcellular distribution of the expressed protein to be determined accurately. Superimposition of the green image and the red image yields a yellow colour when the fluorochromes are colocalized. In this figure a normal peroxisomal protein and a mutated protein deficient in its PTS1 are expressed in normal and pex5 mutant cells (PEX5 encodes the PTS1 import receptor). The normal protein (red) is imported into peroxisomes in the normal cell, as is the peroxisomal marker enzyme catalase (green) (note: red + green = yellow). Neither the normal protein nor the peroxisomal marker is imported into peroxisomes in pex5 mutant cells; instead they both remain in the cytosol. The PTS1-deficient protein is not targeted to peroxisomes in the normal cells but the peroxisomal marker is.

Figure 3. Fluorescence microscopy of cells expressing the reporter protein GFP. When GFP, which does not contain a PTS, is expressed in COS cells it is localized mainly within the cytosol (i.e. a diffuse fluorescence pattern). When a PTS1 is attached to the reporter protein GFP (GFP + PTS1), its distribution becomes punctate, colocalizing with the peroxisomal marker catalase. The arrows highlight colocalization of GFP + PTS1 and catalase.