Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Plant cell or cell line – per se – contains exogenous or...
Reexamination Certificate
1998-06-08
2001-03-06
Bui, Phuong T. (Department: 1638)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Plant cell or cell line, per se, contains exogenous or...
C800S278000, C800S287000, C800S288000, C800S295000, C800S298000, C435S410000, C435S320100, C530S350000, C536S023100, C536S023400, C536S023600, C536S024100
Reexamination Certificate
active
06197588
ABSTRACT:
The present invention relates to plastid inner envelope membrane targeting polypeptides; manufacture and use thereof.
Plastids are organelles present in all plants except blue-green algae, bacteria and fungi. They have various functions and forms but all are bounded by a double membrane envelope. Plastids, including amyloplasts, chromoplasts, leucoplasts and chloroplasts are derived from proplastids in the meristematic cells of plants. The proplastids develop into the various plastid types depending on the cell type and its location in the plant. Amyloplasts, are starch-storage plastids in seeds, tubers and other storage tissues; chromoplasts are brightly coloured carotenoid-containing plastids found in fruits, such as tomatoes and oranges, and in flowers; leucoplasts are colourless plastids found in all other plant cells and often modified for the storage of food; chloroplasts contain the photosynthesis pigments and carry out photosynthesis.
Chloroplasts are complex organelles consisting of six compartments including the outer and inner envelope membranes, the intermembrane space, the stroma, the thylakoid membrane and the thylakoid lumen. Consequently, information is required not only for the targeting of nuclear-encoded proteins to the chloroplast but for sorting to the correct intra-organellar location. The majority of nuclear-encoded chloroplast proteins are synthesised as higher molecular weight precursors with an N-terminal presequence (Keegstra et al., 1989). The presequence directs the protein to the chloroplast surface where it interacts with a proteinaceous receptor in an energy-dependent manner (Cline et al., 1985) and is subsequently translocated across the double envelope membrane. Two approaches, crosslinking of precursors to the import apparatus (Perry and Keegstra, 1994; Wu et al., 1994) and the isolation of translocation complexes (Waegemann and Soll, 1991; Schnell et al., 1994) have enabled the identification of several of the components of the chloroplast import machinery. Maturation of imported protein occurs in the stroma and is catalysed of the stromal processing peptidase (SPP; Oblong and Lamppa, 1992).
Targeting of proteins to the thylakoid membrane and to the thylakoid lumen have been well studied (see de Boer and Weisbeek, 1991) but there have been few studies of envelope membrane targeting.
Most studies have involved the outer envelope membrane and it is not clear what part of the proteins provides the targeting information (Salomon et al., 1990; Li et al., 1990; Ko et al., 1992; Fischer et al., 1994). However, a recently identified outer envelope component of the import machinery does have an N-terminal presequence (Hirsch et al., 1994).
The targeting of three inner envelope membrane proteins has also been studied. These include a 37 kD protein of unknown function (Dreses-Werringloer et al., 1991), the maize Bt1 protein identified as a putative amyloplast membrane metabolite translocator (Sullivan et al., 1991) and the phosphate translocator. The phosphate translocator (PT) is the most abundant chloroplast inner envelope membrane protein (Flügge and Heldt, 1979) and catalyses the export of photosynthate across the inner envelope membrane (Fliege et al., 1978). The mature PT protein is approximately 36 kD and is very hydrophobic containing seven putative membrane-spanning regions. All three inner envelope proteins are synthesised with N-terminal presequences and their import by chloroplasts is both energy-dependent and receptor-mediated (Flügge et al., 1989; Dreses-Werringloer et al., 1991; Li et al., 1992).
The PT precursor proteins from spinach, pea, potato and tobacco have presequences of 72-84 amino acid residues (Flügge et al., 1989; Willey et al., 1991; Schulz et al., 1993; Knight and Gray, 1994) whereas the presequence of the 37 kD precursor protein is 21 amino acid residues (Dreses-Werringloer et al., 1991). The presequences of both proteins are reported to contain features, such as a higher hydroxyl amino acid and a lower arginine content as well as the potential to form an amphiphilic &agr;-helix, which distinguish them from other stromal or thylakoid-targeted proteins. It was suggested that these unusual presequences may be responsible for inner envelope membrane targeting as well as chloroplast targeting (Willey et al., 1991; Dreses-Werringloer et al., 1991).
The present invention has resulted from the identification of residues required for envelope targeting of the phosphate translocator. A number of chimaeric proteins comprising portions of the inner envelope membrane PT fused to the stromal small subunit of ribulose-1,5-bisphosphate carboxylase (SSU Rubisco) were produced and the location of these proteins after their import by isolated chloroplasts was determined. The results indicate that the PT presequence contains only stromal-targeting information and that the mature PT protein is responsible for inner envelope membrane targeting. Further refinement of the chimaeric proteins shows that the N-terminal hydrophobic region of the mature PT protein can direct a stromal protein to the inner envelope membrane demonstrating that targeting information is contained within this region.
The work described herein demonstrates that amino acid residues 24-45 (SEQ ID NO: 1) of the phosphate translocator provide information sufficient for direction of a protein to the inner envelope membrane.
Sequences able to target the chloroplast inner envelope should also target to the envelope of other plastid types in respective cells.
It is an object of the present invention to target the plastid inner envelope membrane of a plant cell.
It is a further object to use this technology to improve the synthesis and storage of starch in plants. Other metabolic processes plastids may also be altered.
Thus according to the method below there is provided a nucleotide sequence encoding a polypeptide comprising amino acid sequence PALTTGFFFFTWYFLNVIFNIL (SEQ ID NO: 1), or an amino acid sequence variant thereof or a derivative thereof. The amino acid sequence SEQ ID NO: 1 is from the pea phosphate translocator. Homologous from other species, and variants and derivatives thereof, are also employed by the present invention. The polypeptide is able to target a plastid inner envelope membrane of a plant cell. Preferably the amino acid sequence is a purified sequence.
The polypeptide sequence may comprise one or more heterologous amino acids joined to the amino acid sequence shown or a variant or derivative thereof. It may retain one or more amino acids from the molecule from which it is derived; for example the polypeptide may comprise one or more amino acids which flank the amino acid sequence SEQ ID NO: 1 in the pea phosphate translocator, or a homologue from another species.
Amino acids joined to the membrane targeting sequence may form a protein domain. Preferably, the sequence of amino acids forms a functional domain. The amino acids may comprise a sequence derived from a membrane transporter or translocator protein, an enzyme, e.g. an enzyme involved in biosynthesis or catabolism, a protein or polypeptide involved in DNA replication or signal transduction including a sequence involved in the control of the growth of cells and hence the development of plant form and function.
The plastid inner envelope is the main permeability barrier of the plastids and therefore targeting transporter or translocator proteins to the envelope may be carried out to change the permeability of the membrane. This may be used to allow the uptake into plastids of compounds that would not normally be transported or to increase uptake or efflux of compounds that are normally transported but at rather slow rates. The transporter proteins (or their genes) may be from any source (animal, plant, fungal, bacterial). One specific example is to increase the permeability of potato tuber amyloplast membranes to glucose 6-phosphate by fusing a gene encoding a glucose 6-phosphate phosphate transporter, e.g. that encoded by the uhpT gene from
Escherichia coli
(Friedrich and Kadner, 1987) to the targ
Gray John Clinton
Knight Jacqueline Sarah
Advanced Technologies (Cambridge) Limited
Bui Phuong T.
Pennie & Edmonds LLP
LandOfFree
Plastid inner envelope membrane targeting polypeptides,... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Plastid inner envelope membrane targeting polypeptides,..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Plastid inner envelope membrane targeting polypeptides,... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2521206