Intestinal aspartate proteases TiCatD and TiCatD2 of the haematophagous bug Triatoma infestans (Reduviidae): sequence characterisation, expression pattern and characterisation of proteolytic activity.

Two aspartate protease encoding complementary deoxyribonucleic acids (cDNA) were characterised from the small intestine (posterior midgut) of Triatoma infestans and the corresponding genes were named TiCatD and TiCatD2. The deduced 390 and 393 amino acid sequences of both enzymes contain two regions characteristic for cathepsin D proteases and the conserved catalytic aspartate residues forming the catalytic dyad, but only TiCatD2 possesses an entire C-terminal proline loop. The amino acid sequences of TiCatD and TiCatD2 show 51-58% similarity to other insect cathepsin D-like proteases and, respectively, 88 and 58% similarity to the aspartate protease ASP25 from T. infestans available in the GenBank database. In phylogenetic analysis, TiCatD and ASP25 clearly separate from cathepsin D-like sequences of other insects, TiCatD2 groups with cathepsin D-like proteases with proline loop. The activity of purified TiCatD and TiCatD2 was highest between pH 2 and 4, respectively, and hence, deviate from the pH values of the lumen of the small intestine, which varied in correlation with the time after feeding between pH 5.2 and 6.7 as determined by means of micro pH electrodes. Both cathepsins, TiCatD and TiCatD2, were purified from the lumen of the small intestine using pepstatin affinity chromatography and identified by nanoLC-ESI-MS/MS analysis as those encoded by the cDNAs. The proteolytic activity of the purified enzymes is highest at pH 3 and the respective genes are expressed in the both regions of the midgut, stomach (anterior midgut) and small intestine, not in the rectum, salivary glands, Malpighian tubules or haemocytes. The temporal expression pattern of both genes in the small intestine after feeding revealed a feeding dependent regulation for TiCatD but not for TiCatD2.

A monoclonal antibody for in vivo detection of peroxisome-associated PTS1 receptor.

PEX5 is a key protein of the peroxisomal protein import machinery. This cycling receptor binds newly synthesized proteins with a peroxisomal targeting signal type 1 in the cytosol and directs them to the peroxisomal membrane. There, PEX5, together with its docking protein, forms a transient membrane-spanning channel that enables cargo-transport across the membrane. Through interaction with other multimeric membrane complexes, the receptor is released from the membrane back to the cytosol. Very little is known about the various conformational states of the receptor during its cycling. Here we report the generation and characterization of a mouse monoclonal antibody that recognizes in vivo primarily the membrane-associated form of the human PTS1 receptor.

The peroxisomal importomer constitutes a large and highly dynamic pore.

The peroxisomal protein import machinery differs fundamentally from known translocons (endoplasmic reticulum, mitochondria, chloroplasts, bacteria) as it allows membrane passage of folded, even oligomerized proteins. However, the mechanistic principles of protein translocation across the peroxisomal membrane remain unknown. There are various models that consider membrane invagination events, vesicle fusion or the existence of large import pores. Current data show that a proteinaceous peroxisomal importomer enables docking of the cytosolic cargo-loaded receptors, cargo translocation and receptor recycling. Remarkably, the cycling import receptor Pex5p changes its topology from a soluble cytosolic form to an integral membrane-bound form. According to the transient pore hypothesis, the membrane-bound receptor is proposed to form the core component of the peroxisomal import pore. Here, we demonstrate that the membrane-associated import receptor Pex5p together with its docking partner Pex14p forms a gated ion-conducting channel which can be opened to a diameter of about 9 nm by the cytosolic receptor-cargo complex. The newly identified pore shows striking dynamics, as expected for an import machinery translocating proteins of variable sizes.

Peroxisomal targeting of PTS2 pre-import complexes in the yeast Saccharomyces cerevisiae.

Posttranslational matrix protein import into peroxisomes uses either one of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. Unlike the PTS1 receptor Pex5p, the PTS2 receptor Pex7p is necessary but not sufficient to target cargo proteins into the peroxisomal matrix and requires coreceptors. Saccharomyces cerevisiae possesses two coreceptors, Pex18p and Pex21p, with a redundant but not a clearly defined function. To gain further insight into the early events of this import pathway, PTS2 pre-import complexes of S. cerevisiae were isolated and characterized by determination of size and protein composition in wild-type and different mutant strains. Mass spectrometric analysis of the cytosolic PTS2 pre-import complex indicates that Fox3p is the only abundant PTS2 protein under oleate growth conditions. Our data strongly suggest that the formation of the ternary cytosolic PTS2 pre-import complex occurs hierarchically. First, Pex7p recognizes cargo proteins through its PTS2 in the cytosol. In a second step, the coreceptor binds to this complex, and finally, this ternary 150 kDa pre-import complex docks at the peroxisomal membrane, where both the PTS1 and the PTS2 import pathways converge. Gel filtration analysis of membrane-bound subcomplexes suggests that Pex13p provides the initial binding partner at the peroxisomal membrane, whereas Pex14p assembles with Pex18p in high-molecular-weight complexes after or during dissociation of the PTS2 receptor.