Related Experiment Video
Updated: Jul 15, 2025
Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays
Published on: June 18, 2016
Loss of mfsd8 alters the secretome during Dictyostelium aggregation
Robert J Huber1, Joshua Gray2, William D Kim2
1Department of Biology, Trent University, Peterborough, Ontario, Canada; Environmental and Life Sciences Graduate Program, Trent University, Peterborough, Ontario, Canada.
Abstract:
Major facilitator superfamily domain-containing protein 8 (MFSD8) is a transmembrane protein that has been reported to function as a lysosomal chloride channel. In humans, homozygous mutations in MFSD8 cause a late-infantile form of neuronal ceroid lipofuscinosis (NCL) called CLN7 disease. In the social amoeba Dictyostelium discoideum, Mfsd8 localizes to cytoplasmic puncta and vesicles, and regulates conserved processes during the organism's life cycle. Here, we used D. discoideum to examine the effect of mfsd8-deficiency on the secretome during the early stages of multicellular development. Mass spectrometry revealed 61 proteins that were differentially released by cells after 4 and 8 h of starvation. Most proteins were present in increased amounts in mfsd8- conditioned buffer compared to WT indicating that loss of mfsd8 deregulates protein secretion and/or causes the release of proteins not normally secreted by WT cells. GO term enrichment analyses showed that many of the proteins aberrantly released by mfsd8- cells localize to compartments and regions of the cell associated with the endo-lysosomal and secretory pathways. Mass spectrometry also revealed proteins previously known to be impacted by the loss of mfsd8 (e.g., cathepsin D), as well as proteins that may underlie mfsd8-deficiency phenotypes during aggregation. Finally, we show that mfsd8-deficiency reduces intracellular proteasome 20S activity due to the abnormal release of at least one proteasomal subunit. Together, this study reveals the impact of mfsd8 loss on the secretome during D. discoideum aggregation and lays the foundation for follow up work that investigates the role of altered protein release in CLN7 disease.
Insights
Loss of Major facilitator superfamily domain-containing protein 8 (MFSD8) in Dictyostelium discoideum disrupts protein secretion, affecting the secretome and proteasome activity. This impacts conserved cellular processes relevant to CLN7 disease.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Major facilitator superfamily domain-containing protein 8 (MFSD8) is a transmembrane protein implicated in lysosomal function.
- Mutations in human MFSD8 cause CLN7 disease, a form of neuronal ceroid lipofuscinosis.
- MFSD8 homologs regulate conserved cellular processes in model organisms like Dictyostelium discoideum.
Purpose of the Study:
- To investigate the impact of mfsd8 deficiency on the secretome during early multicellular development in Dictyostelium discoideum.
- To identify proteins aberrantly released by mfsd8-deficient cells.
- To explore the functional consequences of altered protein release on cellular processes, including proteasome activity.
Main Methods:
- Utilized Dictyostelium discoideum as a model organism to study mfsd8 function.
- Employed mass spectrometry to analyze the secretome of mfsd8-deficient and wild-type cells.
- Performed Gene Ontology (GO) term enrichment analysis on identified proteins.
- Assessed intracellular proteasome 20S activity.
Main Results:
- Mass spectrometry identified 61 differentially released proteins in mfsd8-deficient cells.
- The majority of these proteins were found in increased amounts, indicating deregulated secretion.
- GO analysis revealed enrichment of proteins associated with the endo-lysosomal and secretory pathways.
- mfsd8 deficiency led to reduced intracellular proteasome 20S activity due to abnormal release of proteasomal subunits.
Conclusions:
- Loss of mfsd8 significantly impacts the secretome during Dictyostelium discoideum development.
- Aberrant protein release in mfsd8-deficient cells affects endo-lysosomal and secretory pathways, as well as proteasome activity.
- These findings provide insights into the molecular mechanisms underlying CLN7 disease and highlight the role of altered protein secretion.
