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Title page for ETD etd-11052018-013854


Type of Document Dissertation
Author Takar, Mehmet
Author's Email Address mehmet.takar@vanderbilt.edu
URN etd-11052018-013854
Title Regulation of membrane asymmetry by Golgi P4-ATPases and their interactors
Degree PhD
Department Biological Sciences
Advisory Committee
Advisor Name Title
Brandt Eichman Committee Chair
John D. York Committee Member
Katherine L. Friedman Committee Member
Kathleen L. Gould Committee Member
Todd R. Graham Committee Member
Keywords
  • membrane asymmetry
  • P4-ATPase
  • ATPase
  • transporter
  • phosphatidylserine
  • phosphatidylethanolamine
  • membrane lipid
  • scramblase
Date of Defense 2018-07-12
Availability restrictone
Abstract
Neo1 was discovered in a multi-copy suppressor screen in Saccharomyces cerevisiae to search for factors conferring resistance against an aminoglycoside antibiotic, neomycin (1). Aminoglycoside antibiotics, used to treat bacterial infections by interfering with proofreading during protein synthesis, cause hearing loss in genetically susceptible individuals. It remains unclear why Neo1 overexpression confers resistance to neomycin. Among the yeast P4-ATPases, Neo1 is the only essential P4-ATPase and it has critical roles in COPI-mediated retrograde transport as well as Golgi glycosylation (2,3). In addition, neo1ts mutants display hyperacidification of Golgi membranes and this defect is tightly associated with Neo1’s role in Golgi glycosylation (4). My thesis research studies revealed that Neo1 regulates phosphatidylethanolamine (PE) and phosphatidylserine (PS) asymmetry of the plasma membrane, supporting its proposed function as a phospholipid flippase (5). I provided evidence for the regulation of plasma membrane asymmetry directly by Neo1 rather than indirectly by influencing the activity of Drs2/Dnf P4-ATPases (5). Neo1 also regulates the vacuole fusion machinery and our data suggest this occurs through the enrichment of PE on the cytosolic leaflet of the vacuole membrane (6). The Neo1 homolog Tat-5 in C. elegans was also associated with PE transport activity during embryonic development (7). Together these studies suggest PE is the primary transport substrate for the Neo1/Tat-5/ATP9 orthologs. Unlike Drs2/Dnf P4-ATPases, Neo1 and its human orthologues (Atp9a and Atp9b) do not require a Cdc50-related beta-subunit for either its ER exit nor for its function in vivo (8-11). Evidence others and we have accumulated strongly support the hypothesized role of Neo1 as a phospholipid transporter, but it is still unclear how Neo1 activity is regulated in the Golgi/endosomal membranes.

In the second part of my thesis, we discovered a novel factor antagonizing the Golgi flippase activity called Antagonizes Neo1 Yeast phospholipid flippase (ANY1) in a collaborative study with Charles Boone’s research group. We found that any1∆ completely suppresses the defects that occur from loss of Neo1 and its interaction partners Dop1 and Mon2. Our collaborative study showed that overexpression of Any1 redistributes PS pools from the plasma membrane to internal organelles. On the contrary, overexpression of Any1 only leads to the partial loss of PE asymmetry, but not PS asymmetry. I further identified an essential requirement for a PS flippase activity in the Golgi membranes in the absence of Any1. Serendipitously, I determined that a mutation in transmembrane segment 2 of Neo1 (Neo1[Y222S]) can suppress drs2∆ growth and membrane asymmetry defects. More interestingly, I revealed that the gain of function phenotype is dependent on Any1. Molecular interaction studies performed by immunoprecipitation and proteomics also determined that Neo1 and Any1 physically interact with each other in the Golgi membranes, but it is not clear whether the GOF phenotype of Neo1[Y222S] is due to the loss of Any1 and Neo1 interaction. In summary, Any1 has been introduced as a regulator of lipid landscape in the Golgi/endosomal system.

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