Figure 4.

Characterization of protease-protected PEX5(1–197;C11A): part I. A, RRL containing PEX5(1–197;C11A) (lanes 1 and 2) or PK-treated organelles (Org.) from an in vitro assay programmed with PEX5(1–197;C11A) (lanes 3 and 4) were incubated or not with Genenase I (G I) for 30 min at 23 °C as indicated. Samples were analyzed by SDS-PAGE/Western blotting/autoradiography. The autoradiograph (upper panel) and the corresponding Ponceau S-stained membrane (lower panel) are shown. B, a PK-treated in vitro reaction performed with PEX5(1–197;C11A) was loaded onto the top of a Histodenz gradient and centrifuged. Twelve fractions were then collected from the bottom of the gradient and analyzed by SDS-PAGE/Western blotting/autoradiography. An autoradiograph (upper panel) showing the distribution of PK-resistant ³⁵S-labeled PEX5(1–197;C11A) species and Western blots probed with antibodies directed to PEX14 (peroxisomes), cytochrome c (cyt c; mitochondria), and the retention signal KDEL (endoplasmic reticulum) are presented. Note that PEX14 is converted into a small fragment upon PK digestion (59). C, PK-treated organelles from an in vitro assay programmed with radiolabeled PEX5(1–197;C11A) were disrupted by sonication. Half of the sample was kept on ice (lane T), and the other half was centrifuged to separate membrane (lane P) and soluble (lane S) fractions. Samples were analyzed by SDS-PAGE/Western blotting/autoradiography. The autoradiograph (upper panel) and blots showing the distribution of catalase (a peroxisomal matrix protein) and PEX14 (an intrinsic membrane protein) are presented. D, radiolabeled PEX5(1–197;C11A) was subjected to a PNS-based in vitro assay in the absence (lane 1) or presence (lane 2) of 10 μm NDPEX14. PK-treated organelles were analyzed by SDS-PAGE/autoradiography. The autoradiograph (upper panel) and the corresponding Ponceau S-stained membrane (lower panel) are shown. In C and D, lanes In, RRL containing ³⁵S-labeled PEX5(1–197;C11A). A–D, brackets and arrowheads indicate the PK-cleaved and PK-resistant intact ³⁵S-labeled proteins, respectively. Numbers to the left indicate the molecular mass (kDa) of protein standards.