טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLeboucher Guillaume
SubjectThe Ubiquitin System as a Determinant of Mitochondrial
Function and Morphology
DepartmentDepartment of Biology
Supervisors Professor Michael Glickman
Dr. Allan Weissman
Full Thesis textFull thesis text - English Version


Abstract

The mitochondrion is an essential organelle integral to respiration and apoptosis, and its function is regulated by ongoing fusion and fission events. Two distinct machineries conserved from Saccharomyces cerevisiae to Homo sapiens regulate this dynamic morphology. The fusion of the mitochondrial outer membrane is mediated by GTPase proteins called mitofusins. Knock-outs of mitofusins cause respiration defects in yeast and embryonic lethality in mammals. On the other hand, overexpression of mitofusins results in mitochondrial aggregation. Despite the importance of controlling the levels of mitofusins, regulatory mechanisms responsible for this are still largely unknown. In yeast, the F-box protein regulates Fzo1p levels, however how Mdm30p degrades Fzo1p is unknown. We determined that Mdm30p ubiquitinates the mitofusin Fzo1p, which leads to proteasomal degradation. The Mdm30p F-box motif associates with other components of the Skp1p-Cdc53p-F-box multi-subunit E3 ligase SCF, and this interaction is required for Fzo1p ubiquitination and degradation. Moreover active components of the SCF are required for Fzo1p degradation. Furthermore, we found that the mitochondrial defect observed in an mdm30∆ strain could be partially restored by stimulating the degradation of Fzo1p using the N-end rule pathway, which suggest an additional role for Fzo1p degradation other than regulating its level. Moreover, we establish that Fzo1p degradation depends on the GTPase domain of the mitofusin Fzo1p. GTPase domain-dependent conformational changes in Fzo1p recruit the ubiquitin ligase SCFMdm30p to Fzo1p.

In mammals, we identified that the mitofusin Mfn2 is ubiquitinated and degraded by the proteasome. Moreover, we establish that stress-induced signaling leads to Mfn2 degradation. We identify Mfn2 serine 27 as a site of phosphorylation in response to cellular stress. The mitogen-activated MAP kinase JNK is activated by doxorubicin and is at least partially responsible for Mfn2 phosphorylation. Phosphorylated Mfn2 associates with the HECT domain ubiquitin ligase Huwe1 (HECT, UBA and WWE containing protein 1)/Mule (Mcl-1 ubiquitin ligase E3)/ArfBP1 (Arf binding protein 1)/HectH9. This interaction is mediated at least in part by the BH3 domain of Huwe1 and leads to ubiquitination and proteasomal degradation of Mfn2. In accord with its cellular roles, non-phosphorylatable Mfn2 decreases both mitochondrial fragmentation and cell death in response to doxorubicin. These findings provide a previously unappreciated linkage between cell signaling and mitochondrial function.