Mechanism of Targeting of a Luminal KDEL-containing Protein to the Golgi Complex

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DESCRIPTION (provided by applicant): The objective of this thesis proiect is to define the machinery, mechanism and mode of regulation by which a luminal KDEL- containing protein, ERp44, localizes to the Golgi complex (GC). Understanding how lumenal GC localization is achieved will represent a significant and novel mode of regulatory trafficking and secretory protein quality control. The outcomes of this study will aid in the identification of new therapeutic targets for ER quality control diseases, such as type 2 diabetes. Specifically, we propose three experimental aims. 1. Identify the necessary and sufficient sequence for targeting of ERp44 to the GC. We hypothesize that a targeting sequence exists within ERp44, which overrides its KDEL Endoplasmic Reticulum (ER) retrieval sequence. We propose to identify this sequence by creating truncation, internal deletion, and point mutations, plus chimeric sufficiency constructs and assay their subcellular localization. 2. Identify and characterize the essential machinery for localization of ERp44 to the GC. We will challenge the current model, which asserts ERGIC53 localizes ERp44 to the GC. We postulate receptor machinery exists in the GC that has a higher affinity for a targeting sequence on ERp44 than the KDEL sequence for the KDEL receptor. We will use cross-linking and mutant constructs to define the GC retention protein(s). Live cell fluorescence microscopy photobleaching and biochemistry techniques will be employed to distinguish whether GC localization occurs via retention or retrieval. 3. Determine and characterize the mechanism of regulation of ERp44 localization to the GC. We hypothesize that ERp44 is localized to the ER or GC in a regulated manner. We will investigate a) whether the chaperone function of ERp44 is necessary for GC localization, b) how localization changes with ER stress and c) whether modulation of KDEL-R affects ERp44 localization. Determining how cells regulate the production of secreted proteins is essential to understanding how mutant or inappropriate secretory proteins escape cellular quality control and cause important diseases such as diabetes. Our research will define new machinery that play a key role in promoting or preventing protein quality control.