Functional characterization of the putative lysosomal transporter protein Major facilitator superfamily domain containing 1 (Mfsd1) and its role in sinusoidal obstruction syndrome

• Applicant: Professor Dr. Markus Damme
• Subject Area: Cell Biology ; Biochemistry
• Term: from 2016 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 315767030

• Final Report Year: 2021

Final Report Abstract

Lysosomes are degradative organelles mediating the catalytic breakdown of both intracellular and extracellular substrates. These substrates are processed to lower molecular metabolites by the concerted action of ~60 different acidic enzymes. Low molecular weight metabolites are then exported from the lysosomal lumen to the cytosol by active and selective transport mediated transporter proteins, which facilitate the export between the lumen and the cytosol. MFSD1 is an orphan transporter that we planned to characterize in detail in the project in cell biology experiments, analysis of knockout mice, and biochemical characterization. Our preliminary data implicated that MFSD1 interacts with another lysosomal membrane protein: GLMP. MFSD1 is uncommon for a lysosomal membrane protein, not N-glycosylated. GLMP, in contrast, is highly N-glycosylated. Determining the exact nature of this interaction was another major goal of the project. During the funded project, we figured out by co-immunoprecipitation and FRET-based assays that MFSD1 and GLMP physically interact. The lack of either MFSD1 or GLMP quantitatively regulates the stability of the other interaction partner: In tissues from Mfsd1 knockout mice, GLMP lacks nearly quantitatively, and vice versa in tissues from Glmp knockout mice, MFSD1 is missing nearly completely. The tight genetic interaction is also supported by the strikingly similar phenotype of both knockout mouse strains, which are characterized by a severe liver phenotype characterized by endothelial damage, ultimately leading to fibrosis. In Mouse embryonic fibroblasts from Glmp knockout mice, approximately 10% MFSD1 remains. Interestingly, this remaining MFSD1 is absent from lysosomes but quantitatively found in the Golgi-apparatus. Re-expression of GLMP by transfection restores MFSD1, and MFSD1 is found in lysosomes. Additional experiments suggested that the interaction mediates the stability of the complex of the two proteins and the trafficking from the Golgi-apparatus to lysosomes. Our experiments indicate that the highly N-glycosylated GLMP protects the unglycosylated MFSD1 from lysosomal proteases. Additionally, the interaction of both proteins apparently affects the trafficking of the protein complex from the Golgi-apparatus to lysosomes. Our data provide detailled knowledge on a new couple of an orphan lysosomal transporter, MFSD1, it's accessory subunit GLMP.

Publications

• (2019). "The lysosomal transporter MFSD1 is essential for liver homeostasis and critically depends on its accessory subunit GLMP." eLife 8:e50025
  Massa Lopez, D., M. Thelen, F. Stahl, C. Thiel, A. Linhorst, M. Sylvester, I. Hermanns-Borgmeyer, R. Lullmann-Rauch, W. Eskild, P. Saftig and M. Damme
  
• (2020). "Characterization of the complex of the lysosomal membrane transporter MFSD1 and its accessory subunit GLMP." FASEB journal: 34(11): 14695-14709
  Massa Lopez, D. M., L. Kahlau, K. E. J. Jungnickel, C. Low and M. Damme
  
• (2020). "The lysosomal membrane-export of metabolites and beyond." The FEBS journal
  Rudnik, S. and M. Damme