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  • Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy.

Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy.

Current biology : CB (2017-07-18)
Justin Joachim, Minoo Razi, Delphine Judith, Martina Wirth, Emily Calamita, Vesela Encheva, Brian D Dynlacht, Ambrosius P Snijders, Nicola O'Reilly, Harold B J Jefferies, Sharon A Tooze
초록

Autophagy maintains cellular health and homeostasis during stress by delivering cytosolic material captured by autophagosomes to lysosomes for degradation. Autophagosome formation is complex: initiated by the recruitment of autophagy (Atg) proteins to the formation site, it is sustained by activation of Atg proteins to allow growth and closure of the autophagosome. How Atg proteins are translocated to the forming autophagosome is not fully understood. Transport of the ATG8 family member GABARAP from the centrosome occurs during starvation-induced autophagosome biogenesis, but how centrosomal proteins regulate GABARAP localization is unknown. We show that the centriolar satellite protein PCM1 regulates the recruitment of GABARAP to the pericentriolar material. In addition to residing on the pericentriolar material, GABARAP marks a subtype of PCM1-positive centriolar satellites. GABARAP, but not another ATG8 family member LC3B, binds directly to PCM1 through a canonical LIR motif. Loss of PCM1 results in destabilization of GABARAP, but not LC3B, through proteasomal degradation. GABARAP instability is mediated through the centriolar satellite E3 ligase Mib1, which interacts with GABARAP through its substrate-binding region and promotes K48-linked ubiquitination of GABARAP. Ubiquitination of GABARAP occurs in the N terminus, a domain associated with ATG8-family-specific functions during autophagosome formation, on residues absent in the LC3 family. Furthermore, PCM1-GABARAP-positive centriolar satellites colocalize with forming autophagosomes. PCM1 enhances GABARAP/WIPI2/p62-positive autophagosome formation and flux but has no significant effect on LC3B-positive autophagosome formation. These data suggest a mechanism for how centriolar satellites can specifically regulate an ATG8 ortholog, the centrosomal GABARAP reservoir, and centrosome-autophagosome crosstalk.

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Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Sigma-Aldrich
Anti-γ-Tubulin antibody, Mouse monoclonal, clone GTU-88, ascites fluid
Sigma-Aldrich
2-Chloroacetamide, ≥98%
Sigma-Aldrich
Anti-Ubiquitin Antibody, Lys63-Specific, clone Apu3, rabbit monoclonal, clone Apu3, from rabbit
Millipore
SignalBoostImmunoreaction Enhancer Kit
Sigma-Aldrich
Anti-Vinculin antibody, Mouse monoclonal, clone hVIN-1, purified from hybridoma cell culture
Sigma-Aldrich
Anti-MIB1 (C-terminal) antibody produced in rabbit, ~1.0 mg/mL, affinity isolated antibody, buffered aqueous solution