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Date of Award

8-2024

Document Type

Restricted Thesis: Campus only access

Degree Name

Master of Science in Biology

Department

Biology

First Reader/Committee Chair

Nickerson, Daniel

Abstract

In eukaryotes, Rab GTPases (Rabs) regulate vesicular transport between membrane-bound organelles by cycling between active (GTP-bound) and inactive (GDP-bound) conformations. In GTP-bound active state, a Rab cooperates with effector proteins to regulate transport activities such as membrane tethering, docking and fusion.

Vps21, yeast (S. cerevisiae) ortholog of human Rab5, primarily regulates vesicular transport at endosomes, though it can also localize to and regulate functions at other cellular membranes. The mutant vps21Q66L is a GTP-locked allele that interacts persistently with effector proteins. Using endosomal cargo sorting reporters such as CPY-invertase, we report that there is little to no defect in endosomal transport in vps21Q66L mutants. Interestingly, gas chromatography-mass spectrometry (GC-MS) data and fluorescence microscopy revealed that cells expressing the GTP-locked allele accumulated more lipid droplets (LDs) than wild-type cells. Yeast cells build LDs when nutrients are restricted, suggesting a misregulated response to nutrients. A role for Vps21/Rab5 signaling in regulating LD dynamics represents a new mechanism in understanding lipid metabolism and obesity, though it is still unknown which Vps21 effector protein(s) would contribute to the novel “obese yeast” phenotype.

We intend to identify Vps21 effector proteins that are responsible for LD accumulation through a combination of biotin proximity labeling and mass spectrometry (MS) analysis. We engineered plasmids expressing a fluorophore (mCherry) and biotin ligase mutant (TurboID) fused to the amino- termini of VPS21 and vps21Q66L. We confirmed via microscopy and cargo sorting assays that Vps21 chimeras localize to expected membranes and support Vps21-dependent transport functions. We formulated minimal biotin media to support yeast growth while reducing background biotinylation of native proteins to aid detection of novel Vps21 effectors.

Our TurboID method of proximity labeling biotinylates proteins within the vicinity of Vps21, enabling affinity purification via streptavidin beads of proteins that are covalently tagged with biotin. Streptavidin has a high binding affinity for biotin which allows us to recover biotinylated proteins. Conducting unbiased fishing expeditions via mass spectrometry will allow us to identify Vps21 effector proteins that may be responsible for lipid misregulation. Using chromosomal and plasmid engineering, we have constructed and confirmed via HRP-streptavidin blotting a yeast proteomic query strain expressing a bpl1-temperature sensitive mutation harboring our mCherry-TurboID-Vps21 plasmids. This query strain will allow us to decrease the endogenous biotinylation activity and identify known and novel transient Vps21 effectors related to lipid metabolism which might otherwise be masked by the native biotin ligase machinery.

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