OILSEED ISOCITRATE LYASES LACKING THEIR ESSENTIAL TYPE-1 PEROXISOMAL TARGETING SIGNAL ARE PIGGYBACKED TO GLYOXYSOMES

Isocitrate lyase (IL) is an essential enzyme in the glyoxylate cycle, which is a pathway involved in the mobilization of stored lipids durin g postgerminative growth of oil-rich seedlings. We determined experime ntally the necessary and sufficient peroxisome targeting signals (PTSs ) for cottonseed, oilseed rape, and castor bean ILs in a well-characte rized in vivo import system, namely, suspension-cultured tobacco (Brig ht Yellow) BY-2 cells. Results were obtained by comparing immunofluore scence localizations of wild-type and C-terminal-truncated proteins tr ansiently expressed from cDNAs introduced by microprojectile bombardme nt. The tripeptides ARM-COOH (on cottonseed and castor bean ILs) and S RM-COOH (on oilseed rape IL) were necessary for targeting and actual i mport of these ILs into glyoxysomes, and ARM-COOH was sufficient for r edirecting chloramphenicol acetyltransferase (CAT) from the cytosol in to the glyoxysomes. Surprisingly, IL and CAT subunits without these tr ipeptides were still acquired by glyoxysomes, but only when wild-type IL or CAT-SKL subunits, respectively, were simultaneously expressed in the cells. These results reveal that targeting signal-depleted subuni ts are being piggybacked as multimers to glyoxysomes by association wi th subunits possessing a PTS1. Targeted multimers are then translocate d through membrane pores or channels to the matrix as oligomers or as subunits before reoligomerization in the matrix.