RETRIEVAL PATHWAY IN GOLGI STACK TARGETING

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DESCRIPTION (provided by applicant): Protein targeting mechanisms establish and maintain intracellular compartments. Targeting to the Golgi is an excellent case study as it occurs despite impressive membrane flux through the organelle and it involves subcompartments that mature. Maturation of Golgi cisternae mandates rapid and selective retrieval of Golgi residents from later compartments, presumably by retrieval complexes- cytoplasmic coats selectively enriching for Golgi residents at vesicle bud sites through direct or receptor-mediated interactions with the residents. Thus, what are the retrieval complexes and where do they form? We are pursuing these questions using two Golgi proteins as markers, GPP130 and GP73 that unlike any other known proteins reside in the early Golgi yet redistribute to endosomes upon disruption of lumenal pH and undergo endosome-to-Golgi retrieval upon pH restoration. Our goal is to identify the signals, the signal receptors, and the cytoplasmic coats acting locally in the Golgi and distally in endosomes to mediate retrieval of these proteins. Our progress indicates that the signals are positioned in a coiled-coil lumenal stem region and contain separable elements that confer Golgi and endosomal targeting. The Golgi determinants mediate retrieval from either the Golgi or endosomes and the endosomal determinant diverts a fraction of the protein pool out of the Golgi to endosomes where it undergoes pH-sensitive sorting into retrieval carriers that bypass late endosomes en route back to the Golgi. We will carry out further refinement of the signals and analysis of their role in cycling, and our major effort will be on identification of proteins that functionally interact with these signals and the use of intact, semi-intact, and cell-free assays to identify the cytoplasmic coat components that mediate retrieval sorting of GPP130 and GP73 at the Golgi and in endosomes. For single Golgi proteins that cycle in both the ER/Golgi and TGN/endosome systems, a comparison of requirements for local and long distance cycling may yield significant insights into shared and distinct features in endomembrane targeting.