Oilseed isocitrate lyases lacking their essential type 1 peroxisomal targeting signal are piggybacked to glyoxysomes.

Abstract

Arizona 85287-1 601 lsocitrate lyase (IL) is an essential enzyme in the glyoxylate cycle, which is a pathway involved in the mobilization of stored lipids during postgerminative growth of oil-rich seedlings. We determined experimentally the necessary and sufficient peroxisome targeting signals (PTSs) for cottonseed, oilseed rape, and castor bean ILs in a well-characterized in vivo import system, namely, suspension-cultured tobacco (Bright Yellow) BY-2 cells. Results were obtained by comparing immunofluorescence localizations of wild-type and C-terminal-truncated proteins transiently expressed from cDNAs introduced by microprojectile bombardment. The tripeptides ARM-COOH (on cottonseed and castor bean ILs) and SRM-COOH (on oilseed rape IL) were necessary for targeting and actual import of these ILs into glyoxysomes, and ARM-COOH was sufficient for redirecting chloramphenicol acetyltransferase (CAT) from the cytosol into the glyoxysomes. Surprisingly, IL and CAT subunits without these tripeptides 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 subunits are being piggybacked as multimers to glyoxysomes by association with subunits possessing a PTSl. Targeted multimers are then translocated through membrane pores or channels to the matrix as oligomers or as subunits before reoligomerization in the matrix.

Disrupting Autophagy Restores Peroxisome Function to anArabidopsis lon2Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

“…Is it possible that all three mechanisms are in play? Peroxisomal protein import is unusual in that matrix proteins can be imported as fully folded, oligomeric complexes (McNew and Goodman, 1994;Lee et al, 1997). Rather than fully degrading matrix proteins, perhaps LON2 processes or disaggregates matrix proteins, receptor complexes, or receptor-matrix protein complexes to allow their ubiquitin-dependent retrotranslocation out of the organelle.…”

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

“…Other PTS2-containing proteins dimerize or form higher multimers, suggesting that this may be a more general observation (Mathieu et al, 1994;Guex et al, 1995;Lee et al, 1997;Flynn et al, 1998;Chudzik et al, 2000). However, thiolase can be imported as a heterodimer when one subunit possesses a PTS2 and the other does not, which suggests that PTS2 dimerization is not required for import (Glover et al, 1994;Flynn et al, 1998).…”

Novel Targeting Signals Mediate the Sorting of Different Isoforms of the Tail-Anchored Membrane Protein Cytochrome b5 to Either Endoplasmic Reticulum or Mitochondria

“…These plasmids were then digested each with NheI and XbaI, and the resulting DNA fragments were gel purified and ligated into XbaI-digested pRTL2-mycX. The pRTL2-mycX plasmid is a modified version of the plant expression vector pRTL2DN/S (Lee et al, 1997) that includes sequences encoding an initiator Met, Gly linkers, and the myc-epitope tag (underlined, MGEQKLISEEDLG-;Fritze and Anderson, 2000) followed by an in-frame XbaI site that allows for the convenient ligation of passenger coding sequences. The construction of other yeast expression vectors (pYES2.1) containing myc-tagged versions of the four tung Cb5 ORFs was performed using PCR with the appropriate forward and reverse primers and the pRTL2-myc-Cb5 plasmids as template DNA.…”