Chloroplasts are the subcellular organelles of plants that convert light energy from the sun into biologically usable form and generate the atmospheric oxygen without which life as we know it on earth today could not exist. The vital chemical reaction catalyzed by the enzymes in the chloroplasts is the combining of carbon dioxide with water and energy to produce glucose plus molecular oxygen. The glucose is then used by the plant (and any animal that eats the plant) as an energy-rich food that can be utilized either directly or indirectly to fuel metabolism. The compartmentation of chloroplast proteins within the multiply membrane-bounded chloroplast is intrinsically critical to its function. Since most of the chloroplast proteins, including the light-harvesting enzymes, are manufactured in the cytoplasm, the cells have evolved a complex mechanism for transporting these proteins to their proper places in the chloroplast. In higher plants, the chloroplasts contain two envelope membranes, and although not all the details are worked out, there is already a good deal known about the protein translocation machineries in higher plant chloroplasts. However, in certain algal cells, the chloroplasts contain three or even four envelope membranes. Far less is understood about how proteins are translocated into these so-called complex chloroplasts. Understanding the biogenesis of complex chloroplasts has important implications for evolution as well as for our understanding of fundamental mechanisms of eukaryotic protein targetting in general. This project has as its long term goal the understanding of protein targetting and translocation to the complex chloroplasts of the unicellular alga Euglena.
Eukaryotic cells target and translocate proteins to specific subcellular compartments in ways that differ from one compartment to another in their details; however, there are some general themes, such as the presence of specific targeting sequences in the protein, that are common to all these processes. Generally, chloroplast protein precursors have an N-terminal presequence, the transit peptide, containing information required for post-translational import into chloroplasts. Precursors to both stromal and thylakoid proteins contain a functionally similar transit peptide that targets the precursor to the stroma through a general import pathway. Protein translocation into the endoplasmic reticulum (ER) is usually co-translational and dependent upon an N-terminal presequence, the signal peptide. Integral membrane proteins contain hydrophobic stop transfer membrane anchor sequences that stop translocation, anchoring the protein within the membrane. Proteins destined for other intracellular compartments are transported in vesicles from the ER to the Golgi apparatus, sorted in the Golgi apparatus and packaged in transport vesicles for transfer to their final intracellular location.
Euglena chloroplasts contain 3 envelope membranes. Stromal and thylakoid proteins are transported in vesicles as integral membrane proteins from the ER to the Golgi apparatus to the outermost plastid envelope membrane, rather than directly from the cytoplasm to chloroplast. All Euglena chloroplast protein presequences are 140 amino acids in length with a similar structure composed of a N-terminal signal peptide and a second hydrophobic domain 60 amino acids from the signal peptidase cleavage site. This second hydrophobic domain anchors the protein in the membrane with the presequence N-terminus inside the microsomal lumen and the C-terminus on the cytoplasmic membrane face. A Euglena precursor was imported into pea chloroplasts indicating that the Euglena presequence contains a transit peptide recognized by the higher plant import machinery. The Euglena intermediate and inner envelope should contain proteins homologous to components of the plant envelope import apparatus that interact with the transit peptide. Euglena plastid envelope proteins have not been characterized. The most novel targeting region of the Euglena presequence, the Golgi to chloroplast targeting sequence, is also unidentified.
The first objective of this project is to develop procedures to isolate the Euglena chloroplast envelope and identify Euglena chloroplast envelope proteins that are part of the protein import apparatus. Advantage will be taken of the fact that two plant envelope proteins, Toc 34 and Toc159, that interact with the transit peptide are GTP binding proteins. This biochemical property will be used to isolate the Euglena Toc homologues. Peptide sequence will be obtained and used to design degenerate primers for cDNA isolation by PCR. Isolation of the cDNAs represents the first step toward characterization of the Euglena envelope import apparatus. The second objective is to develop a Euglena transformation system that can be used to identify the Golgi to chloroplast targeting domain and the chloroplast import (transit peptide) domains in the Euglena chloroplast protein precursor presequence. The Sh ble gene encoding zeocin resistance fused to the 5' and 3' untranslated regions of the Euglena LHCPII gene will be used as a positive selectable marker allowing optimization of transformation conditions by electroporation or biolistic transformation. Cells will be co-transformed with the Sh ble gene and precursor-GFP fusion protein presequence deletion constructs. Zeocin resistant cells will be screened by fluorescence microscopy for GFP expression and confocal microscopy will be used for an initial determination of intracellular localization. The precursor-GFP fusion proteins will contain the Euglena stromal polyprotein processing peptidase cleavage site so that upon chloroplast import, GFP will be released from the fusion protein providing a simple biochemical assay (western blotting) to confirm import. The third objective is to identify the ancestral function of the Euglena presequence domains by using confocal microscopy to determine the intracellular localization of precursor-GFP fusion protein presequence deletion constructs in mammalian cells.
