The subcellular localization of nutrient transporters, growth factor receptors, cell adhesion molecules, SNARE proteins and many ion channels and pumps rely on dynamic trafficking itineraries between the Golgi, plasma membrane and endosomal system. Localization of these proteins is regulated at the critical decision point of maintaining an internal distribution between the Golgi and endosomes, returning back to the plasma membrane, or being sent to lysosomes for degradation. Many pathogenic processes, particularly those that cause neurodegenerative disease, disrupt these normal protein trafficking itineraries. However, the mechanisms driving many discrete steps of these transport pathways are unknown, and thus the molecular basis of many neurodegenerative diseases and best course of treatment remains a critically important unanswered question. Preliminary studies suggest a new function for an old vesicle coat protein - COPI - in transporting ubiquitinated cargo from the early endosome to the Golgi so they can recycle back to the plasma membrane. COPI is known to associate with early endosomes, although its function at this organelle is not known. In addition, the role of ubiquitin in endocytosis and sorting proteins into the multivesicular body pathway for degradation in lysosomes is well characterized, but how ubiquitination could rescue a protein from a lysosomal fate is unclear.
The first aim i s to fully define the function of COPI and ubiquitin in recycling a SNARE protein from early endosomes to the Golgi. This COPI-dependent pathway requires a phosphatidylserine (PS) flippase (Drs2) and an F-box protein (Rcy1) that appears to act in an SCF-independent mechanism.
The second aim will test the hypothesis that COPI recruitment to early endosomes requires Arf-GTP, an ARFGAP, ubiquitinated cargo and PS concentrated on the cytosolic leaflet by the flippase. The primary function of Rcy1 and the ubiquitin ligase acting at this step will be characterized. Preliminary studies suggest a second, backup pathway exists to recycle the ubiquitinated SNARE from late endosomes using a sorting nexin 4 (Snx4) complexes. A genetic screen identified at least two new protein transport factors that appear to function with the Snx4 complex.
The third aim i s to define the function of these new factors and determine if they are involved in cargo selection at the late endosomes. Successful completion of this work could ultimately impact the diagnosis and treatment of neurodegenerative diseases in humans.

Public Health Relevance

Protein trafficking between the plasma membrane, endosomes and Golgi is crucial for normal cellular functions and is disrupted in Alzheimer's disease, Parkinson's disease, cardiovascular disease, cancer and a number of rare genetic disorders. The proposed studies will provide fundamental insight into how proteins travel between cellular compartments and may lead to better diagnoses and treatments for these diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Membrane Biology and Protein Processing Study Section (MBPP)
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Flicker, Paula F
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Vanderbilt University Medical Center
Schools of Arts and Sciences
United States
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Xu, Peng; Hankins, Hannah M; MacDonald, Chris et al. (2017) COPI mediates recycling of an exocytic SNARE by recognition of a ubiquitin sorting signal. Elife 6: