Identification and developmental expression of the mitochondrial phosphate transport protein gene from the spruce budworm, Choristoneura fumiferana

https://doi.org/10.1016/S0965-1748(98)00055-1Get rights and content

Abstract

Phosphate transport protein (PTP) is a mitochondrial inner membrane protein responsible for the translocation of inorganic phosphate into the mitochondrial matrix. A full length cDNA clone encoding the PTP was isolated from the spruce budworm, Choristoneura fumiferana. The deduced amino acid sequence of the longest ORF of CfPTP cDNA showed high similarity with the amino acid sequences of PTPs cloned from several species. Phylogenetic tree analysis indicated that CfPTP occupied an intermediate position between vertebrates on the one side and yeast and nematodes on the other side. Studies on the developmental expression of CfPTP mRNA showed that higher levels of mRNA were present during the feeding and growing stages than during molting periods.

Introduction

Phosphate transport protein (PTP) is a major protein of the inner mitochondrial membrane and is responsible for the translocation of inorganic phosphate into the mitochondrial matrix for oxidative phosphorylation in energy metabolism of cells (LaNoue and Schoolwerth, 1979). PTP has been isolated and purified to homogeneity from bovine heart (Kolbe et al., 1984) and rat liver mitochondria (Kaplan et al., 1986) by chromatographic methods. The protein has also been functionally reconstituted in proteoliposomes (Kaplan et al., 1986, Stappen and Kramer, 1993). The PTPs are 33 to 34 kDa in size and they most likely act as a dimer in the inner membrane of mitochondria (Kramer, 1996). The sequence of an amino-terminal fragment (Kolbe and Wohlrab, 1985) and partial sequence (Aquila et al., 1987) of the bovine heart PTP have been determined by chemical protein sequence analysis. The complete amino acid sequence of the PTP was deduced from the nucleotide sequence of cDNA clones from bovine heart (Runswick et al., 1987) and rat liver (Ferreira et al., 1989), whereas the PTP sequences from yeast (Phelps et al., 1991) and human heart (Dolce et al., 1994) were from genomic clones. The bovine and human PTP genes have a similar structure consisting of nine exons separated by eight introns (Palmieri et al., 1993, Dolce et al., 1994). The PTP belongs to a superfamily of mitochondrial carrier proteins, including ADP/ATP carrier (AAC), uncoupling protein (UCP) and oxoglutarate carrier (Runswick et al., 1987, Runswick et al., 1990, Dietmeier et al., 1993, Palmieri et al., 1993). All these carrier proteins are not only structurally homologous to each other, but also have a similar targeting mechanism as well as a submitochondrial location (Dietmeier et al., 1993). They are located in the inner membrane of mitochondria and contain three-fold repeated sequences that are conserved. Each repeated element contains approximately 100 amino acids and is composed of two hydrophobic membrane-spanning α-helices linked by an extensive hydrophillic domain (Runswick et al., 1987, Aquila et al., 1987).

Several models on the topography of the PTP in the mitochondrial membrane have been described. Aquila et al. (1985), Aquila et al. (1987)suggested that it was made up of six transmembrane α-helices and a β-strand spanning across the inner mitochondrial membrane. The N-terminal end of the protein was located in the matrix side of the membrane and the C-terminal end was at the cytosol side. Based on the studies on PTP from rat liver mitochondria, Ferreira et al. (1989)suggested that it consisted of six hydrophobic α-helices connected by five extra-membrane hydrophillic loops that were membrane-spanning with both the N-terminal and C-terminal regions of the peptide chain being oriented towards the matrix side of the inner membrane. Capobianco et al. (1991)and Palmieri et al. (1993)proposed a similar model in which six membrane-spanning α-helices cross the membrane but the amino and the carboxyl termini were both located at the cytosol side of the membrane. This topology is the same for other mitochondrial carrier proteins.

Several reviews on mitochondrial PTP have been published with emphasis on the transport system (LaNoue and Schoolwerth, 1979, Kramer, 1996), molecular aspects (Wohlrab, 1986), and comparison of PTP with other mitochondrial carriers (Aquila et al., 1987), but few studies have been reported on the developmental expression of the PTP mRNA with the exception of Dolce et al. (1996)who investigated the tissue-specific expression of two isoforms of the mitochondrial PTP in bovine tissues.

In the present paper, we describe the isolation, sequencing and characterization of a full length PTP cDNA clone (CfPTP) from a lepidopteran insect, C. fumiferana. We compare CfPTP with the PTPs from other species and discuss their phylogenetic relationships. In addition, we studied the developmental expression of the CfPTP mRNA and discuss its role in development of C. fumiferana.

Section snippets

Experimental animals

Spruce budworm (Choristoneura fumiferana Clem., Lepidoptera: Tortricidae) eggs were collected within one hour after oviposition and maintained at 22°C and 70% relative humidity. Under these conditions, the eggs hatched in eight days. After five days, the first instar larvae molted into second instars and entered diapause. The hibernacula of the diapausing larvae in cheese cloth sheets were stored at 2°C for 20–25 weeks to satisfy the diapause requirement. At the end of diapause, the larvae were

Identification and sequencing of CfPTP cDNA clone

The cDNA inserts from ten clones randomly picked from the cDNA library were sequenced on both ends. The deduced amino acid sequence of one of the cDNA clones showed high similarities with the deduced amino acid sequences of mitochondrial phosphate transport proteins (PTP) cloned from several species (see below). Because of its high similarity with PTPs, this cDNA clone was designated as C. fumiferana phosphate transport protein (CfPTP). The cDNA was then sequenced completely on both strands.

The

Discussion

In this study, we report the isolation, sequencing of cDNA and developmental expression of mRNA for CfPTP. The identity of the cloned cDNA was confirmed by comparing the cDNA sequence with the sequences of PTPs from other species. Since the cDNA clone is 1767 nucleotides in length and cDNA probe binds to a 1.8-kb mRNA (Fig. 4, Fig. 5, Fig. 6, Fig. 7), we believe that this is a full length cDNA clone. This is the only second invertebrate and first insect PTP cDNA cloned.

PTPs of mammalian

Acknowledgements

This research is supported in part by a grant from the Natural Science and Engineering Research Council to K.G. Davey and by the National Biotechnology Strategy Fund, and the Science and Technology Opportunities Fund of Canadian Forest Service to S.R. Palli.

References (34)

Cited by (0)

View full text