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Mutational analyses of a type 2 peroxisomal targeting signal that is capable of directing oligomeric protein import into tobacco BY‐2 glyoxysomes
Author(s) -
Flynn C. Robb,
Mullen Robert T.,
Trelease Richard N.
Publication year - 1998
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1998.00344.x
Subject(s) - peroxisomal targeting signal , peroxisome , glyoxysome , biochemistry , biology , signal peptide , chloramphenicol acetyltransferase , microbody , nicotiana tabacum , peptide sequence , microbiology and biotechnology , gene , gene expression , promoter
Summary In this study of the type 2 peroxisomal targeting signal (PTS2) pathway, we examined the apparent discontinuity and conservation of residues within the PTS2 nonapeptide and demonstrated that this topogenic signal is capable of directing heteromultimeric protein import in plant cells. Based on cumulative data showing that at least 26 unique, putative PTS2 nonapeptides occur within 12 diverse peroxisomal‐destined proteins, the current (‐R/K‐L/V/I‐X 5 ‐H/Q‐L/A‐) as well as the original (‐R‐L‐X 5 ‐H/Q‐L‐) PTS2 motif appear to be oversimplified. To assess the functionality of residues within the motif, rat liver thiolase (rthio) and various chimeric chloramphenicol acetyltransferase (CAT) proteins were expressed transiently in suspension‐cultured tobacco (Nicotiana tabacumL.) cv Bright Yellow cells (BY‐2), and their subcellular location was determined by immunofluorescence microscopy. Hemagglutinin (HA)‐epitope‐tagged‐CAT subunits, lacking a PTS2 (CAT‐HA), were ‘piggybacked’ into glyoxysomes by PTS2‐bearing CAT subunits (rthio‐CAT), whereas signal‐depleted CAT‐HA subunits that were modified to prevent oligomerization did not import into glyoxysomes. These results provided direct evidence that signal‐depleted subunits imported into peroxisomes were targeted to the organelle as oligomers (heteromers) by a PTS2. Mutational analysis of residues within PTS2 nonapeptides revealed that a number of amino acid substitutions were capable of maintaining targeting function. Furthermore, functionality of residues within the PTS2 nonapeptide did not appear to require a context‐specific environment conferred by adjacent residues. These results collectively suggest that the functional PTS2 is not solely defined as a sequence‐specific motif, i.e. ‐R/K‐X 6 ‐H/Q‐A/L/F‐, but defined also by its structural motif that is dependent upon the physiochemical properties of residues within the nonapeptide.