A Single Peroxisomal Targeting Signal Mediates Matrix Protein Import in Diatoms

Abstract

Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

“…To corroborate the transmission electron microscopy evidence that punctate NR resides in the peroxisome ( Figures 1C and 1D), NR was colocalized with a known peroxisomal matrix protein, 3-ketoacyl-CoA thiolase (KAT), which was identified in P. tricornutum by Gonzalez et al (2011). YFP-NR and CFP-KAT constructs were each inserted into regulatory cassettes controlled by the NR promoter (ProNR) and NR terminator (TermNR) (Poulsen and Kröger, 2005).…”

Biology of a widespread uncultivated archaeon that contributes to carbon fixation in the subsurface

“…The substitution medium consisted of acetone in combination with 0.2% (w/v) osmium tetroxide, 0.25% (w/v) uranyl acetate and 5% (v/v) water. The substitution program including washing steps and the following epon embedding, thin sectioning and post staining was carried out as described previously 61 . For these samples, microscopy was performed on a JEOL JEM-2100 (JEOL, Tokyo, Japan) also operated at 120 kV and equipped with a 2 k  2 k fast scan CCD camera F214 combined with EM Menu4 (TVIPS GmbH, Gauting, Gemany).…”

Evidence for glycoprotein transport into complex plastids

“…To substitute the water and enhance the contrast of the samples, a mixture consisting of 0.2% OsO 4 , 0.25% uranyl acetate, and 5% (vol/vol) H 2 O in acetone was used. The substitution program, the following washing steps, Epon embedding, ultrathin sectioning, and immunogold labeling were performed as described previously (45). Labeling of the GFP fusion proteins was performed with a primary antibody against GFP (goat-αGFP; Rockland, diluted 1:1,000) and a secondary 10-nm gold-coupled rabbit-α-goat IgG (dilution 1:20) with subsequent poststaining as described previously (46).…”