Mahogunin regulates fusion between amphisomes/MVBs and lysosomes via ubiquitination of TSG101

Abstract

Aberrant metabolic forms of the prion protein (PrP), membrane-associated CtmPrP and cytosolic (cyPrP) interact with the cytosolic ubiquitin E3 ligase, Mahogunin Ring Finger-1 (MGRN1) and affect lysosomes. MGRN1 also interacts with and ubiquitinates TSG101, an ESCRT-I protein, involved in endocytosis. We report that MGRN1 modulates macroautophagy. In cultured cells, functional depletion of MGRN1 or overexpression of CtmPrP and cyPrP blocks autophagosome–lysosome fusion, alleviates the autophagic flux and its degradative competence. Concurrently, the degradation of cargo from the endo-lysosomal pathway is also affected. This is significant because catalytic inactivation of MGRN1 alleviates fusion of lysosomes with either autophagosomes (via amphisomes) or late endosomes (either direct or mediated through amphisomes), without drastically perturbing maturation of late endosomes, generation of amphisomes or lysosomal proteolytic activity. The compromised lysosomal fusion events are rescued by overexpression of TSG101 and/or its monoubiquitination in the presence of MGRN1. Thus, for the first time we elucidate that MGRN1 simultaneously modulates both autophagy and heterophagy via ubiquitin-mediated post-translational modification of TSG101.

Ubiquitin mediated regulation of the E3 ligase GP78 by Mahogunin in trans affects mitochondrial homeostasis

“…Maturation of late endosomes, generation of amphisomes and lysosomal proteolytic activity also does not get perturbed in these conditions (Majumder and Chakrabarti, 2015). Hence, we sought to confirm that the mitochondrial changes observed in MGRN1-depleted cells were due to GP78, and cells were co-transfected with different MGRN1 and GP78 constructs in the indicated combinations.…”

Endosomal sorting complexes required for ESCRTing cells toward death during neurogenesis, neurodevelopment and neurodegeneration

“…A recent study from our laboratory has shown that the ubiquitination of TSG101 is important in the fusion of lysosomes with autophagosomes and amphisomes; this maintains the intracellular autophagic flux and its degradative competence. Thus, loss of ubiquitinated TSG101 leads to build up of toxic cargo in cells because of impaired lysosomal fusion . Furthermore, loss of ubiquitinated functional TSG101 effectuates accumulation of unfolded proteins in the lumen of endoplasmic reticulum (ER), triggering ER stress; prolonged ER stress leads to apoptosis in cultured SHSY5Y (human neuroblastoma) and HeLa (human cervical cancer) cells .…”

“…Homozygous knockout animal for the ESCRT‐I protein, TSG101, is embryonically lethal . Depletion of TSG101 in cellular systems show enlarged lysosomes enriched with LC3 without perturbation of LAMP‐1 or CTSD trafficking and localization, suggesting its involvement in autophagic pathway . Furthermore, TSG101 dysfunction causes accumulation of autophagosomes due to inhibition of fusion of autophagosomes/amphisomes with lysosomes .…”

“…30,52,75 Recent work from our laboratory has demonstrated that ubiquitination of the ESCRT-I protein, TSG101, by MGRN1 helps in fusion of autophagosome/amphisome with lysosomes. 41 We have identified a mechanism by which presence of Ctm PrP and cyPrP results in impaired degradation by the macroautophagic pathway in cell culture systems and transgenic mice. 41,43 More recently, another study identified that tamoxifeninduced conditional deletion of Tsg101 from mature oligodendroglia leads to severe spongiform encephalopathy similar to that observed in Mgrn1 null mice but with a more rapid onset.…”

“…41 We have identified a mechanism by which presence of Ctm PrP and cyPrP results in impaired degradation by the macroautophagic pathway in cell culture systems and transgenic mice. 41,43 More recently, another study identified that tamoxifeninduced conditional deletion of Tsg101 from mature oligodendroglia leads to severe spongiform encephalopathy similar to that observed in Mgrn1 null mice but with a more rapid onset. 76 Here it was suggested that the biogenesis of vacuoles in central nervous system was a result of defective MVB formation-again implicating TSG101 in prion related neurodegeneration.…”