The characterization of AAA-ATPase Afg1 in Saccharomyces cerevisiae

Zoe Keese Author

04/02/2021 Added

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In this study, we attempted to identify a role of yeast Afg1 in response to other homeostatic insults including heavy metals, osmotic stress, calcium overload, mitochondrial depolarization, respiration, and blockage of potassium channels. We then extended our investigation to a potential role of Afg1 in MQC via regulation of autophagy, mitophagy and lipophagy.

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[00:00:00.670]My name is Zoe Keese,
[00:00:01.850]and I'm a sophomore biochemistry major.
[00:00:04.360]I will be presenting my UCARE Project,
[00:00:06.360]addressing the characterization
[00:00:07.840]of the mitochondrial AAA enzyme Afg1
[00:00:10.940]in Saccharomyces cerevisiae.
[00:00:14.280]The research was performed
[00:00:15.460]in Professor Oleh Khalimonchuk's lab
[00:00:17.380]at the Department of Biochemistry and Redox Biology Center.
[00:00:22.370]Mitochondria, also known as the powerhouse of the cell,
[00:00:25.660]are involved in a range of vital cellular processes,
[00:00:28.750]such as respiration, cofactor synthesis,
[00:00:31.350]lipid homeostasis, and more.
[00:00:34.840]Such complex machinery requires
[00:00:36.740]an efficient maintenance system
[00:00:38.300]represented by a variety of factors and mechanisms
[00:00:41.130]known as mitochondrial quality control or MQC.
[00:00:46.360]Professor Khalimonchuk's lab
[00:00:47.830]is focused on protein quality control systems comprised
[00:00:51.020]of evolutionary conserved proteases and chaperones.
[00:00:54.550]Despite the functional importance of the PGC,
[00:00:57.500]the identities and roles of many of its components
[00:00:59.870]are not well-understood.
[00:01:01.560]One of such components
[00:01:02.730]is the matrix-localized AAA+ ATPase Afg1.
[00:01:08.420]Afg1 is highly evolutionarily conserved
[00:01:11.190]from yeasts to humans.
[00:01:13.030]It is a member of classical AAA+ ATPase family
[00:01:17.160]and functions primarily as a chaperone.
[00:01:20.930]Using growth spot assay,
[00:01:22.450]we attempted to identify a role of yeast Afg1
[00:01:25.310]in response to various homeostatic insults.
[00:01:28.310]Pre-grown overnight cultures were serially diluted
[00:01:31.100]and plated on YPD plates containing stress factors,
[00:01:34.580]including heavy metals, osmotic stress, calcium overload,
[00:01:38.160]mitochondrial depolarization, respiration,
[00:01:41.230]and blockage of potassium channels.
[00:01:45.120]Yeast cells lacking Afg1 grew well
[00:01:47.320]in the presence of non-fermentable carbon sources
[00:01:50.080]which require robust mitochondrial bioenergetics function.
[00:01:54.410]Also, there were no growth defects observed
[00:01:56.700]in the presence of the oxidative phosphorylation inhibitor.
[00:02:00.930]Similarly, growth of the Afg1 deficient cells
[00:02:03.910]was not affected by calcium overload, osmotic stress,
[00:02:07.250]or potassium channels blockage.
[00:02:10.240]Finally, we have not observed
[00:02:11.850]any growth defects of Afg1 mutant cells
[00:02:14.260]in the presence of various metals.
[00:02:17.570]We have then investigated if Afg1 is involved in autophagy.
[00:02:21.530]We have used previously developed GFP-Atg8
[00:02:24.580]to measure the autophagy flux.
[00:02:26.830]If GFP is fused to the N-terminus of Atg8, LC3,
[00:02:31.330]it remains attached to the protein
[00:02:33.110]until vacuolar, lysosomal, delivery.
[00:02:35.850]Atg8 is degraded relatively rapidly compared to GFP,
[00:02:39.800]so the generation of free GFP
[00:02:41.500]in the vacuolar/lysosomal lumen
[00:02:43.370]can be used to monitor autophagy activity.
[00:02:47.620]Using this assay,
[00:02:48.800]we have measured autophagic flux in Afg1-deficient cells
[00:02:52.340]using Atg1 mutant defective and mitophagy as our control.
[00:02:56.580]It appears that Afg1 is not involved
[00:02:58.640]in autophagy regulation.
[00:03:01.380]Similar assay with mitophagy marker
[00:03:03.540]Om45 tagged with GFP was performed.
[00:03:06.920]Once more, we have not observed any mitophagy deficiency
[00:03:10.230]in the Afg1 mutant compared to the wild type strain.
[00:03:15.400]We have also measured lipophagic flux
[00:03:17.510]using Erg6-GFP processing assay.
[00:03:20.710]Lack of Afg1 did not affect lipophagy
[00:03:23.250]under conditions tested.
[00:03:25.760]To summarize, Afg1 is an evolutionary conserved AAA-ATPase.
[00:03:31.040]Afg1 is dispensable for protection
[00:03:33.320]against various homeostatic insults in the yeast model.
[00:03:36.770]And Afg1 is not required
[00:03:38.470]for autophagy, mitophagy, and lipophagy.
[00:03:43.320]We will further continue the search
[00:03:44.820]for potential Afg1 functions and its targets.
[00:03:48.030]Previous proteomics analysis
[00:03:49.860]of Afg1 hexahistidine co-immunoprecipitation fractions
[00:03:53.890]identify the set
[00:03:54.890]of potential interacting partners of this factor,
[00:03:57.720]including metabolic enzymes, Oac1, and Ach1.
[00:04:01.830]These results are consistent
[00:04:03.130]with previously observed Afg1-depleted cells' inability
[00:04:06.910]to quickly adjust in response to metabolic challenges,
[00:04:10.210]such as nutrient carbon source exchange.
[00:04:13.620]We will obtain HA epitope tag variant of Oac1
[00:04:17.730]and will generate Ach1 variant
[00:04:19.830]with the C-terminal FLAG-tag.
[00:04:22.220]The constructs will be used to validate these interactions
[00:04:25.010]in reciprocal co-immunoprecipitation experiments
[00:04:28.230]with wild type
[00:04:29.120]and catalytically impaired E206Q mutant variants
[00:04:33.420]of hexahistidine tag AFg1.
[00:04:36.660]In addition, steady state levels of these enzymes
[00:04:39.600]in young and chronologically aged wild type
[00:04:42.020]and Afg1 mutant cells will be tested.
[00:04:46.160]Thank you for your time.

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The characterization of AAA-ATPase Afg1 in Saccharomyces cerevisiae

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