A novel dynein-type AAA+ protein with peroxisomal targeting signal type 2

Peroxisomal matrix proteins are imported into peroxisomes in a process mediated by peroxisomal targeting signal (PTS) type 1 and 2. The PTS2 proteins are imported into peroxisomes after binding with Pex7p. Niwa et al. (A newly isolated Pex7-binding, atypical PTS2 protein P7BP2 is a novel dynein-type AAA+ protein. J Biochem 2018;164:437–447) identified a novel Pex7p-binding protein in CHO cells and characterized the subcellular distribution and molecular properties of the human homologue, ‘P7BP2’. Interestingly, P7BP2 possesses PTS2 at the NH2 terminal and six putative AAA+ domains. Another group has suggested that the protein also possesses mitochondrial targeting signal at the NH2 terminal. In fact, the P7BP2 expressed in mammalian cells is targeted to both peroxisomes and mitochondria. The purified protein from Sf9 cells is a monomer and has a disc-like ring structure, suggesting that P7BP2 is a novel dynein-type AAA+ family protein. The protein expressed in insect cells exhibits ATPase activity. P7BP2 localizes to peroxisomes and mitochondria, and has a common function related to dynein-type ATPases in both organelles.

Allostery between two binding sites in the ion channel subunit TRIP8b confers binding specificity to HCN channels

Tetratricopeptide repeat (TPR) domains are ubiquitous structural motifs that mediate protein–protein interactions. For example, the TPR domains in the peroxisomal import receptor PEX5 enable binding to a range of type 1 peroxisomal targeting signal motifs. A homolog of PEX5, tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b), binds to and functions as an auxiliary subunit of hyperpolarization-activated cyclic nucleotide (HCN)–gated channels. Given the similarity between TRIP8b and PEX5, this difference in function raises the question of what mechanism accounts for their binding specificity. In this report, we found that the cyclic nucleotide–binding domain and the C terminus of the HCN channel are critical for conferring specificity to TRIP8b binding. We show that TRIP8b binds the HCN cyclic nucleotide–binding domain through a 37-residue domain and the HCN C terminus through the TPR domains. Using a combination of fluorescence polarization– and co-immunoprecipitation–based assays, we establish that binding at either site increases affinity at the other. Thus, allosteric coupling of the TRIP8b TPR domains both promotes binding to HCN channels and limits binding to type 1 peroxisomal targeting signal substrates. These results raise the possibility that other TPR domains may be similarly influenced by allosteric mechanisms as a general feature of protein–protein interactions.

In SilicoAnalysis ofArabidopsis thalianaPeroxisomal 6-Phosphogluconate Dehydrogenase

NADPH, whose regeneration is critical for reductive biosynthesis and detoxification pathways, is an essential component in cell redox homeostasis. Peroxisomes are subcellular organelles with a complex biochemical machinery involved in signaling and stress processes by molecules such as hydrogen peroxide (H2O2) and nitric oxide (NO). NADPH is required by several peroxisomal enzymes involved inβ-oxidation, NO, and glutathione (GSH) generation. Plants have various NADPH-generating dehydrogenases, one of which is 6-phosphogluconate dehydrogenase (6PGDH). Arabidopsis contains three6PGDHgenes that probably are encoded for cytosolic, chloroplastic/mitochondrial, and peroxisomal isozymes, although their specific functions remain largely unknown. This study focuses on thein silicoanalysis of the biochemical characteristics and gene expression of peroxisomal 6PGDH (p6PGDH) with the aim of understanding its potential function in the peroxisomal NADPH-recycling system. The data show that a group of plant 6PGDHs contains an archetypal type 1 peroxisomal targeting signal (PTS), whilein silicogene expression analysis using affymetrix microarray data suggests that Arabidopsis p6PGDH appears to be mainly involved in xenobiotic response, growth, and developmental processes.

Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals

Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes.