CONSERVATIVE AMINO-ACID SUBSTITUTIONS OF THE C-TERMINAL TRIPEPTIDE (ALA-ARG-MET) ON COTTONSEED ISOCITRATE LYASE PRESERVE IMPORT IN-VIVO INTO MAMMALIAN-CELL PEROXISOMES

The purpose of this research was twofold, a) to directly demonstrate i mport in vivo of a native plant peroxisomal protein into peroxisomes o f transiently transfected mammalian cells, and b) to identify the targ eting signal and amino acid substitutions thereof which preserve trans location of this plant protein into these peroxisomes. The protein sel ected for study was cottonseed isocitrate lyase (ICL), a glyoxylate cy cle enzyme which participates in storage oil mobilization in oilseed c otyledons. Cultured mammalian cells were selected as the import system because of previous success by others with transient transfections an d import of heterologous (not plant, however) proteins, and because ne ither a plant in vitro or transient in vivo import system was establis hed. Optimized transient transfections of cultured CV-1 monkey kidney, mouse L, HeLa, and CHO cells resulted in punctate, anti-cottonseed-IC L-dependent immunofluorescent patterns. Colocalization in a CVH Px110 cell line of ICL with either endogenous catalase or with stably expres sed CAT-PMP20/AKL (chloramphenicol acetyltransferase with a C-terminal -appended 12 amino acids ending with Ala-Lys Leu) demonstrated targeti ng of ICL to peroxisomes. Direct evidence for translocation of ICL int o CHO cell peroxisomes was obtained from digitonin permeabilization ex periments. The necessity of the C-terminal tetrapeptide, KARM-COOH, wa s demonstrated in CHO and CV-1 cells when removal of this tetrapetide (leaving ICL-WA-COOH) abolished import into peroxisomes. This result i s in general agreement with Olsen et al. (The Plant Cell 5, 941-952 (1 993)) who demonstrated that the 37 C-terminal amino acids of oilseed r ape ICL were necessary for import in vivo in transgenic plants. The fi ndings of Behari and Baker (J. Biol, Chem. 268, 7315-7322 (1993)), how ever, indicate that the C-terminal portion of castor bean ICL is dispe nsible for import in vitro. Single or multiple conservative amino acid substitutions at each position of the C-terminal tripeptide of native cottonseed ICL (S for A, K for R, L for M, SK for AR, SKL for ARM) pr eserved import of the enzyme in vivo into CHO cell peroxisomes. The de monstrated targeting and translocation of plant ICL and C-terminal mod ifications thereof into mammalian cell peroxisomes provide important a dditional evidence for evolutionary conservation of peroxisome import machinery, especially relative to the PTS1 sequence.