Targeting of glyoxysomal proteins to peroxisomes in leaves and roots of a higher plant.

Abstract

Higher plants possess several classes of peroxisomes that are present at distinct developmental stages and serve different metabolic roles. To investigate the cellular processes that regulate developmental transitions of peroxisomal function, we analyzed the targeting of glyoxysomal proteins to leaf-type and root peroxisomes. We transferred genes encoding the glyoxysome-specific enzymes isocitrate lyase (IL) and malate synthase into Arabidopsis plants and showed, in cell fractionation and immunogold localization experiments, that the glyoxysomal proteins were imported into leaf-type and root peroxisomes. We next defined the sequences that target IL to peroxisomes and asked whether the same targeting determinant is recognized by different classes of the organelle. By localizing deletion and fusion derivatives of IL, we showed that the polypeptide's carboxyl terminus is both necessary for its transport to peroxisomes and sufficient to redirect a passenger protein from the cytosol to both glyoxysomes and leaf-type peroxisomes. Thus, glyoxysomal proteins are transported into several classes of peroxisomes using a common targeting determinant, suggesting that protein import does not play a regulatory role in determining a peroxisome's function. Rather, the specific metabolic role of a peroxisome appears to be determined primarily by processes that regulate the synthesis and/or stability of its constituent proteins. These processes are specified by the differentiated state of the cells in which the organelles are found.

Matrix proteins are inefficiently imported into Arabidopsis peroxisomes lacking the receptor-docking peroxin PEX14

“…After transfer, the membranes were air dried, blocked for 1 h in 8% non-fat dry milk in TBS-T (blocking buffer), and incubated overnight at 4°C with primary antibodies diluted in blocking buffer: rabbit α-GFP (1:300 dilution, BD Biosciences 8372-2), rabbit α-ICL (1:2,000 dilution, Maeshima et al 1988), rabbit α-MLS (1:25,000 dilution, Olsen et al 1993), rabbit α-PEX5 (1:100 dilution, Zolman and Bartel 2004), rabbit α-PEX6 (1:1,000 dilution, Ratzel et al 2011), rabbit α-PEX7 (1:800 dilution, Ramón and Bartel 2010), rabbit α-PEX13 (1:500 dilution, Mano et al 2006), rabbit α-PEX14 (1:2,500 dilution, Lingard and Bartel 2009), rabbit α-PMDH2 (1:2,000 dilution, Pracharoenwattana et al 2007), rabbit α-thiolase (PED1 isoform, 1:10,000 dilution, Lingard et al 2009), mouse α-complex V subunit α (1:2,000 dilution, MitoScience MS507), or mouse α-HSC70 (1:4,000–1:20,000 dilution, StressGen Bioreagents SPA-817), followed by a 1–4 h incubation with horseradish peroxidase-conjugated α-rabbit or α-mouse secondary antibody (Santa Cruz Biotechnology). Horseradish peroxidase was visualized by incubation with LumiGlo reagent (Cell Signaling Technology, Danvers, MA).…”

Pexophagy and peroxisomal protein turnover in plants

“…Extracts from 6-day-old light-grown seedlings of the indicated genotypes were processed for immunoblotting in duplicate, and membranes were serially probed with antibodies recognizing the peroxisome matrix proteins thiolase [13] or malate dehydrogenase [PMDH; 82], shown in the upper panel, or malate synthase [MLS; 83], shown in the lower panel. Thiolase and PMDH are PTS2 proteins synthesized as precursors (p) in the cytosol and cleaved to mature forms (m) lacking the PTS2 region in the peroxisome.…”

Transport of proteins in eukaryotic cells: more questions ahead

“…Plant cells possess several classes of peroxisomes, and chimeric proteins fused to PTS-like sequences are correctly directed into the various types of peroxisomes [194,266]. Studies aimed at identifying peroxisomal OM proteins [43] and developing an in vivo peroxisome import assay in plants [10] are initial steps toward understanding the specific molecular events related to import into this organelle.…”