Processing the Polyprotein Precursor to Euglena LHCPII

University Of Memphis, Memphis TN

Investigators

Abstract

96-30817 Schwartzbach Most of the proteins in chloroplasts are nuclear-encoded, synthesized in the cytoplasm, and imported post-translationally into the chloroplast. Precursors of chloroplast proteins contain topogenic information for targeting to and import into chloroplasts. Precursors of Euglena light harvesting chlorophyll a/b binding protein of photosystem 11 (pLHCP11) and of the small subunit of ribulose bisphosphate carboxylase (pSSU) are polyproteins that are composed of mature polypeptides covalently linked by a conserved decapeptide. In vivo pulse chase experiments demonstrated that pLHCP11 and pSSU are transported as integral membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus prior to chloroplast localization rather than being imported directly into the chloroplast. Once the polyproteins are in the chloroplast they are processed by an endoproteolytic polyprotein processing peptidase. PLHCP11 and pSSU each contain a signal peptide for targeting to the ER and a stop-transfer signal C-terminal of the signal peptide. Studies using canine microsomes and deletion constructs demonstrated that the stop-transfer signal anchors the protein in the membrane with the N-terminus in the microsomal lumen and the C-terminus in the cytoplasm. Previous work described the import and processing pathway of Euglena chloroplast proteins. The objective of this research is to understand the molecular mechanism of this novel trafficking pathway for Euglena chloroplast proteins. This will be achieved by developing an in vitro system that reconstitutes the Golgi apparatus to chloroplast transport event. This system should allow for the biochemical characterization of this step as well as for the identification of components that are essential for this process. This includes the isolation and characterization of the polyprotein processing peptidase(s). %%% Most of the proteins in chloroplasts are nuclear-encoded, synthesized in the cytoplasm, and imported post-translationally int o the chloroplast. Precursors of chloroplast proteins contain topogenic information for targeting to and import into chloroplasts. The objective of this research is to investigate a novel trafficking pathway for nuclear-encoded chloroplast proteins to Euglena chloroplasts. The precursors of chloroplast proteins are first targeted to the endoplasmic reticulum, transported to the Golgi apparatus and are then transported to the chloroplast. Nothing is known about the molecular mechanism of this novel trafficking pathway for precursors of chloroplast proteins to the chloroplast. ***