Targeting and Function of Golgi Membrane Proteins

Johns Hopkins University, Baltimore MD

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Abstract

DESCRIPTION (provided by applicant): The Golgi complex is a ubiquitous eukaryotic organelle that plays a central role in post-translational processing and sorting of newly synthesized proteins and lipids. One of the best-studied functions of the Golgi complex is oligosaccharide processing, which is precisely controlled by expression and localization of glycosyltransferases and glycosidases. Glycosylation patterns change during development, and aberrant glycosylation patterns may contribute to metastasis of tumor cells. The sorting function of the Golgi complex is also critical, since protein and lipid mistargeting can lead to disease. The Golgi complex has an unusual structure consisting of sets of stacked cisternal membranes, which is likely to be important for proper function. Peripheral Golgi membrane proteins with large coiled-coil domains called golgins have been implicated in Golgi structure and function. Golgin-160 is the focus of this proposal. Although little is known regarding the function of golgin-160, it appears to play a critical role in Golgi structure. Previous work has shown that golgin-160 is cleaved by caspases during apoptosis (programmed cell death), promoting Golgi disassembly. In addition, certain cleavage fragments of the protein are transported to the nucleus. Cleavage of golgin-160 may be an early event induced by cellular stress signals that are sensed at Golgi membranes. Transduction of certain types of signals may thus be another, unappreciated function of the Golgi complex. The hypotheses that will be tested by the proposed research are that golgin-160 is essential for normal Golgi structure and function, that stress signals are transmitted through golgin-160 by caspase cleavage, and that golgin-160 cleavage fragments are transported to the nucleus to regulate gene expression for stress repair. The specific aims are to: (1) Determine the role of golgin-160 in maintaining Golgi structure and function. This will be accomplished by determining the precise localization of golgin-160, assessing the effects of knocking down its expression using small interfering RNA, and identifying its binding partners. (2) Determine how stress signals are sensed at Golgi membranes. Stimuli that activate caspase-2 at the Golgi complex will be identified, and the mechanism by which dominant negative mutants of golgin-160block downstream signaling will be determined. (3) Determine the potential functions of cleavage fragments of golgin-160 that enter the nucleus by assessing how they are produced and translocated, and if they regulate gene expression.