Elucidating the role of selective Golgi remodeling in cellular homeostasis - Project Summary/ Abstract: Mammalian cells can tune spatial distribution of proteins and remodel the proteome in response to a myriad of cellular stress conditions through protein production and degradation. Macroautophagy (autophagy), an evolutionarily conserved degradation pathway, delivers cellular macromolecules to the lysosome for recycling1,2. Autophagy in response to nutrient stress has long been considered to result in non-specific capture of cytoplasmic contents within autophagosomes to generate biosynthetic building blocks3–6. However, recent work revealed a pathway that enables the selective encapsulation and delivery of Golgi membrane proteins to the lysosome for recycling upon nutrient stress (Golgiphagy), mediated by two Golgi-resident multipass transmembrane proteins (YIPF3 and YIPF4)7. Thus, autophagy entails layers of selective organelle turnover (principally Golgi and ER) rather than simply random cytosol encapsulation. Beyond nutrient stress, YIPF3/4- mediated Golgiphagy also selectively remodels the Golgi proteome during in vitro neuronal differentiation,7,8, suggesting a broad role for YIPF3/4 in regulating Golgi homeostasis. Despite this finding, the molecular mechanism(s) that enable the selective degradation of Golgi proteins by autophagy, and the functional consequences of Golgi remodeling on neuronal homeostasis remains completely unknown. The proposed research aims to understand the molecular mechanisms that underpin Golgiphagy, and the impact of autophagy, and other quality control pathways, on neuronal Golgi homeostasis. AIM 1 will elucidate molecular mechanisms of YIPF3/4 mediated selective Golgiphagy and provide training in the biochemical techniques necessary to purify, reconstitute, and dissect mechanisms of membrane proteins. AIM 2 will create a 3D super-resolution Golgi protein map to uncover the impact of autophagy on establishing neuronal Golgi protein organization and develop tools to rigorously investigate Golgi biology in neurons. Finally, the independent phase of this award, AIM 3, will uncover Golgi quality control pathways using systematic assays, followed by mechanistic dissection of the underlying mechanisms, using the knowledge and tools developed during the mentored phase of this award. Understanding the pathways of Golgi quality control in neurons will provide insights into the connection between Golgi dysregulation and neurodegenerative disease. The mentored phase of this award (AIM 1-2) will provide both biological and technical skills that will be essential for the PI’s success as an independent investigator, including AIM 3 of this application. The mentored phase of this award provides the opportunity to work with leaders in the fields of Golgi biology and neurobiology to acquire the training needed to achieve the PI’s long-term goal of becoming an independent investigator at the interface of both fields. Together, the proposed research, mentorship team, and resources provided for career development at Harvard Medical School will provide the training needed to achieve the defined career goals.
