Dataset Information

Spatial regulation of UBXD8 and p97/VCP controls ATGL-mediated lipid droplet turnover.

ABSTRACT: UBXD8 is a membrane-embedded recruitment factor for the p97/VCP segregase that has been previously linked to endoplasmic reticulum (ER)-associated degradation and to the control of triacylglycerol synthesis in the ER. UBXD8 also has been identified as a component of cytoplasmic lipid droplets (LDs), but neither the mechanisms that control its trafficking between the ER and LDs nor its functions in the latter organelle have been investigated previously. Here we report that association of UBXD8 with the ER-resident rhomboid pseudoprotease UBAC2 specifically restricts trafficking of UBXD8 to LDs, and that the steady-state partitioning of UBXD8 between the ER and LDs can be experimentally manipulated by controlling the relative expression of these two proteins. We exploit this interaction to show that UBXD8-mediated recruitment of p97/VCP to LDs increases LD size by inhibiting the activity of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triacylglycerol hydrolysis. Our findings show that UBXD8 binds directly to ATGL and promotes dissociation of its endogenous coactivator, CGI-58. These data indicate that UBXD8 and p97/VCP play central integrative roles in cellular energy homeostasis.

SUBMITTER: Olzmann JA

PROVIDER: S-EPMC3557085 | biostudies-other | 2013 Jan

REPOSITORIES: biostudies-other

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Spatial regulation of UBXD8 and p97/VCP controls ATGL-mediated lipid droplet turnover.

Olzmann James A JA Richter Caleb M CM Kopito Ron R RR

Proceedings of the National Academy of Sciences of the United States of America 20130107 4

UBXD8 is a membrane-embedded recruitment factor for the p97/VCP segregase that has been previously linked to endoplasmic reticulum (ER)-associated degradation and to the control of triacylglycerol synthesis in the ER. UBXD8 also has been identified as a component of cytoplasmic lipid droplets (LDs), but neither the mechanisms that control its trafficking between the ER and LDs nor its functions in the latter organelle have been investigated previously. Here we report that association of UBXD8 wi ...[more]

PMID: 23297223

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Project description:Choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine in cholinergic neurons, and mutations of this enzyme are linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related mutation, V18M, reduces ChAT enzyme activity and cellular protein levels, and is located within a highly-conserved N-terminal proline-rich motif at residues 14PKLPVPP20. We showed previously that disruption of this proline-rich motif by either proline-to-alanine mutation (P17A/P19A) or mutation of residue Val18 (V18M) enhances ubiquitination and degradation of these mutant ChAT proteins expressed in cholinergic SN56 cells by an unknown mechanism. In this study, using proximity-dependent biotin identification (BioID), co-immunoprecipitation and in situ proximity-ligation assay (PLA), we identified the heat shock proteins (HSPs) HSC/HSP70 and HSP90 as novel ChAT protein-interactors. These molecular chaperones are well-known for promoting the folding and stabilization of cellular proteins. Thus, we found that inhibition of HSPs by treatment of cells with either the HSC/HSP70 inhibitors 2-phenylethynesulfonamide (PES) or VER-155008, or the HSP90 inhibitor 17-AAG reduced cellular ChAT activity and solubility, and enhanced the ubiquitination and proteasome-dependent loss of ChAT protein. Importantly, the effects of HSP inhibition were greater for mutant ChAT proteins (P17A/P19A-ChAT and CMS-related V18M- and A513T-ChAT) compared to wild-type ChAT. HSPs can promote ubiquitination and degradation of terminally misfolded proteins through cooperative interaction with the E3 ubiquitin ligase CHIP/Stub1, and while we show that ChAT interacts with CHIP in situ, siRNA-mediated knock-down of CHIP had no effect on either wild-type or mutant ChAT protein levels. However, inhibition of the endoplasmic reticulum (ER)- and HSP-associated co-chaperone p97/VCP prevented degradation of ubiquitinated ChAT. Together, these results identify novel mechanisms for the functional regulation of wild-type and CMS-related mutant ChAT by pro-stabilizing HSPs and the pro-degradative co-chaperone p97/VCP that may have broader implications for ChAT function during cellular stress and disease.

Project description:Maintaining mitochondrial function is essential for neuronal survival and offers protection against neurodegeneration. Ubiquitin-mediated, proteasome-dependent protein degradation in the form of outer mitochondrial membrane associated degradation (OMMAD) was shown to play roles in maintenance of mitochondria on the level of proteostasis, but also mitophagy and cell death. Recently, the AAA-ATPase p97/VCP/Cdc48 was recognized as part of OMMAD acting as retrotranslocase of ubiquitinated mitochondrial proteins for proteasomal degradation. Thus, p97 likely plays a major role in mitochondrial maintenance. Support for this notion comes from mitochondrial dysfunction associated with amyotrophic lateral sclerosis and hereditary inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) caused by p97 mutation. Using SH-SY5Y cells stably expressing p97 or dominant-negative p97(QQ) treated with mitochondrial toxins rotenone, 6-OHDA, or A?-peptide as model for neuronal cells suffering from mitochondrial dysfunction, we found mitochondrial fragmentation under normal and stress conditions was significantly increased upon inactivation of p97. Furthermore, inactivation of p97 resulted in loss of mitochondrial membrane potential and increased production of reactive oxygen species (ROS). Under additional stress conditions, loss of mitochondrial membrane potential and increased ROS production was even more pronounced. Loss of mitochondrial fidelity upon inactivation of p97 was likely due to disturbed maintenance of mitochondrial proteostasis as the employed treatments neither induced mitophagy nor cell death. This was supported by the accumulation of oxidatively-damaged proteins on mitochondria in response to p97 inactivation. Dysfunction of p97 under normal and stress conditions in neuron-like cells severely impacts mitochondrial function, thus supporting for the first time a role for p97 as a major component of mitochondrial proteostasis.

Dataset Information

Spatial regulation of UBXD8 and p97/VCP controls ATGL-mediated lipid droplet turnover.

Publications

Spatial regulation of UBXD8 and p97/VCP controls ATGL-mediated lipid droplet turnover.

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