UCLA_Wohlschlegel_Lab - xfan_CIA_PRM

Iron-Regulated Assembly of the Cytosolic Iron-Sulfur Cluster Biogenesis Machinery
Data License: CC BY 4.0 | ProteomeXchange: PXD033557 | doi: https://doi.org/10.6069/41va-8n81
  • Organism: Homo sapiens
  • Instrument: Orbitrap Fusion Lumos
  • SpikeIn: No
  • Keywords: PRM, iron-sulfur cluster, protein interactions
  • Lab head: James Wohlschlegel Submitter: Xiaorui Fan
The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that NUBP1 (NBP35), CIAO3 (NARFL) and CIA substrates associate with NUBP2 (CFD1), a component of the CIA scaffold complex. We also show that NUBP2 weakly associates with the CIA targeting complex (MMS19, CIAO1, CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.
Experiment Description
Please see description in the manuscript
Sample Description
Fig. 1C: Two technical replicates of HEK293 lysate with different input amount Fig. 2B: Two biological replicates of anti-HA immunoprecipitate against 3HA-3FLAG-CIAO3 under DFO condition (2D1, 2D2) and FAC condition (2F1, 2F2) Fig. 4B to 4D: Two biological replicates of anti-HA immunoprecipitate against 3HA-3FLAG-CIAO3 wild-type and mutants. 293FlpIn background (1,6); C71S (2,7); C190S/C395S (3,8); C71S/C190S/C395S (4,9); WT (5,10)
Created on 4/28/22, 4:03 PM
Clustergrammer Heatmap
Fig4BtoD_centroid_CIA_PRM_v3_MS2only_unique_protein_5640_9606_20190515_v1.1_2022-04-27_17-08-05.sky.zip2022-04-28 16:03:1010272818310
Fig1C_centroid_CIA_PRM_v5_MS2only_unique_protein_5640_9606_20210910_v1.1_2022-04-27_15-57-33.sky.zip2022-04-28 16:03:1015464829710
Fig2B_centroid_CIA_PRM_v4_MS2only_unique_protein_5640_9606_IOP1_20200214_v1.1_2022-04-27_15-36-21.sky.zip2022-04-28 16:03:10616161354