Genetics of secretion in yeast

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Principal Investigator: PETER Jay NOVICK
Organization: UNIVERSITY OF CALIFORNIA, SAN DIEGO
Fiscal Year: 2024
Award: $465,901
Funding agency: National Institute of General Medical Sciences

Project Summary
The endoplasmic reticulum (ER) undergoes autophagic degradation in response to the accumulation of
aggregated proteins within its lumen or in response to starvation. We have proposed that in yeast, actin
assembly at sites of contact between the cortical ER (cER) and endocytic pits is required to displace elements
of the cER from their association with the plasma membrane so that they can interact with the autophagosome
assembly machinery near the vacuole.
 To test our model, we will determine if the requirement in cER-phagy for actin assembly at endocytic
pits can be bypassed by deleting all six genes encoding ER-plasma membrane tethers (DTether). If the role of
actin assembly is to push the cER away from the plasma membrane, the DTether mutations should bypass the
need for actin assembly in ER-phagy. We will ask if the bypass of actin assembly is specific to cER-phagy or if
actin-related defects in other selective autophagy pathways, such as nucleophagy, mitophagy, and pexophagy
are also bypassed. As an additional test we will ask if an artificial ER-plasma membrane tether can block cER-
phagy in both wild type and the DTether strain. Extensive parallels between ER-phagy in yeast and mammalian
cells suggest a conserved mechanism. A long-term goal will be to use siRNA knock downs of actin assembly
components in U2OS cells to assess their roles in ER-phagy.
 We will order all known cER-phagy requirements with respect to the displacement of the cER from the
plasma membrane. Our recent data suggests that a vesicle coat adaptor protein associates with a selective
ER-phagy receptor prior to the displacement of cER from the plasma membrane. We will develop a
bimolecular fluorescence complementation assay to visualize the dynamics of this ER-phagy intermediate in
live cells. We will also order the displacement of the cER from the plasma membrane with respect to the super
assembly of Atg40 oligomers, a process required for packaging of ER fragments into autophagosomes. We will
explore a novel fluorescence complementation assay for super assembly of Atg40 oligomers that will allow
visualization in live cells. In total our studies will define the pathway by which the ER is degraded by
autophagy. This pathway plays a key role in a variety of human diseases including neurodegenerative
disorders and forms of diabetes.

Terms: <Actin-Activated ATPase><Actins><Adaptor Protein><Adaptor Protein Gene><Adaptor Signaling Protein><Adaptor Signaling Protein Gene><Affect><Assay><Autophagocytosis><Autophagosome><Bioassay><Biological Assay><Bypass><COPII-Coated Vesicles><Cell Body><Cell membrane><Cells><Coated vesicle><Complement><Complement Proteins><Complex><Crnlp protein><Cytoplasmic Domain><Cytoplasmic Membrane><Cytoplasmic Tail><Data><Defect><Degenerative Neurologic Disorders><Diabetes Mellitus><Elements><Endocytosis><Endoplasmic Reticulum><Endosomes><Ergastoplasm><Event><Fluorescence><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic defect><Goals><Impairment><Link><Lysosomes><Mammalian Cell><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Microscopy><Modeling><Molecular><Mutation><Myosin ATPase><Myosin Adenosine Triphosphatase><Myosin Adenosinetriphosphatase><Myosins><Nervous System Degenerative Diseases><Neural Degenerative Diseases><Neural degenerative Disorders><Neurodegenerative Diseases><Neurodegenerative Disorders><Neurologic Degenerative Conditions><Pathway interactions><Physical condensation><Plasma Membrane><Play><Polymers><Process><Proteins><Receptor Protein><Receptosomes><Role><Short interfering RNA><Site><Small Interfering RNA><Starvation><Testing><Vacuole><Visualization><Yeasts><aberrant folded protein><aberrant folded proteins><abnormal folded protein><abnormal folded proteins><adapter protein><autophagy><complementation><condensation><coronin><coronin protein><degenerative diseases of motor and sensory neurons><degenerative neurological diseases><diabetes><gene testing><gene-based testing><genetic testing><genome mutation><human disease><inhibitor><insoluble aggregate><knock-down><knockdown><membrane-organizing extension spike protein><misfolded protein><misfolded proteins><moesin><mutant><neurodegenerative illness><novel><pathway><plasmalemma><polymer><polymeric><polymerization><protein aggregate><protein aggregation><proteotoxic protein><proteotoxin><receptor><recruit><response><scaffold><scaffolding><siRNA><social role>