Mt-Keima detects PINK1-PRKN mitophagy with greater sensitivity than mito-QC.

PINK1 and PRKN, which cause Parkinson disease when mutated, form a quality control mitophagy pathway that is well-characterized in cultured cells. The extent to which the PINK1-PRKN pathway contributes to mitophagy , however, is controversial. This is due in large part to conflicting results from studies using one of two mitophagy reporters: mt-Keima or mito-QC.

Fis1 deficiencies differentially affect mitochondrial quality in skeletal muscle.

Mitochondrial dynamics and mitophagy are important aspects of mitochondrial quality control, and are linked to neurodegenerative diseases and muscular diseases. Fis1, a protein on the mitochondrial outer membrane, is thought to mediate mitochondrial fission. However, Fis1 null worms and mammalian cells only display mild fission defects but show aberrant mitophagy.

Parkin and PINK1 mitigate STING-induced inflammation.

Although serum from patients with Parkinson's disease contains elevated levels of numerous pro-inflammatory cytokines including IL-6, TNF, IL-1β, and IFNγ, whether inflammation contributes to or is a consequence of neuronal loss remains unknown. Mutations in parkin, an E3 ubiquitin ligase, and PINK1, a ubiquitin kinase, cause early onset Parkinson's disease. Both PINK1 and parkin function within the same biochemical pathway and remove damaged mitochondria from cells in culture and in animal models via mitophagy, a selective form of autophagy.

The erlin2 T65I mutation inhibits erlin1/2 complex-mediated inositol 1,4,5-trisphosphate receptor ubiquitination and phosphatidylinositol 3-phosphate binding.

The erlin1/2 complex is a ∼2-MDa endoplasmic reticulum membrane-located ensemble of the ∼40-kDa type II membrane proteins erlin1 and erlin2. The best defined function of this complex is to mediate the ubiquitination of activated inositol 1,4,5-trisphosphate receptors (IPRs) and their subsequent degradation. However, it remains unclear how mutations of the erlin1/2 complex affect its cellular function and cause cellular dysfunction and diseases such as hereditary spastic paraplegia.

Vps13D Encodes a Ubiquitin-Binding Protein that Is Required for the Regulation of Mitochondrial Size and Clearance.

The clearance of mitochondria by autophagy, mitophagy, is important for cell and organism health [1], and known to be regulated by ubiquitin. During Drosophila intestine development, cells undergo a dramatic reduction in cell size and clearance of mitochondria that depends on autophagy, the E1 ubiquitin-activating enzyme Uba1, and ubiquitin [2]. Here we screen a collection of putative ubiquitin-binding domain-encoding genes for cell size reduction and autophagy phenotypes.

Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria.

Selective autophagy of damaged mitochondria requires autophagy receptors optineurin (OPTN), NDP52 (CALCOCO2), TAX1BP1, and p62 (SQSTM1) linking ubiquitinated cargo to autophagic membranes. By using quantitative proteomics, we show that Tank-binding kinase 1 (TBK1) phosphorylates all four receptors on several autophagy-relevant sites, including the ubiquitin- and LC3-binding domains of OPTN and p62/SQSTM1 as well as the SKICH domains of NDP52 and TAX1BP1. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and kinase activation on mitochondria.

The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.

Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, the ubiquitin kinase PINK1 phosphorylates ubiquitin to activate the ubiquitin ligase parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors. Using genome editing to knockout five autophagy receptors in HeLa cells, here we show that two receptors previously linked to xenophagy, NDP52 and optineurin, are the primary receptors for PINK1- and parkin-mediated mitophagy.

A Point Mutation in the Ubiquitin Ligase RNF170 That Causes Autosomal Dominant Sensory Ataxia Destabilizes the Protein and Impairs Inositol 1,4,5-Trisphosphate Receptor-mediated Ca2+ Signaling.

RNF170 is an endoplasmic reticulum membrane ubiquitin ligase that contributes to the ubiquitination of activated inositol 1,4,5-trisphosphate (IP3) receptors, and also, when point mutated (arginine to cysteine at position 199), causes autosomal dominant sensory ataxia (ADSA), a disease characterized by neurodegeneration in the posterior columns of the spinal cord. Here we demonstrate that this point mutation inhibits RNF170 expression and signaling via IP3 receptors. Inhibited expression of mutant RNF170 was seen in cells expressing exogenous RNF170 constructs and in ADSA lymphoblasts, and appears to result from enhanced RNF170 autoubiquitination and proteasomal degradation.

High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy.

An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson's disease. Recent studies of the Parkinson's disease associated genes PINK1 (ref. 2) and parkin (PARK2, ref.

RNF170 protein, an endoplasmic reticulum membrane ubiquitin ligase, mediates inositol 1,4,5-trisphosphate receptor ubiquitination and degradation.

Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum membrane calcium channels that, upon activation, are degraded via the ubiquitin-proteasome pathway. While searching for novel mediators of IP(3) receptor processing, we discovered that RNF170, an uncharacterized RING domain-containing protein, associates rapidly with activated IP(3) receptors. RNF170 is predicted to have three membrane-spanning helices, is localized to the ER membrane, and possesses ubiquitin ligase activity.

Activated inositol 1,4,5-trisphosphate receptors are modified by homogeneous Lys-48- and Lys-63-linked ubiquitin chains, but only Lys-48-linked chains are required for degradation.

Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are large, ubiquitously expressed, endoplasmic reticulum membrane proteins that form tetrameric IP(3) and Ca(2+)-gated Ca(2+) channels. Endogenous IP(3)Rs provide very appealing tools for studying the ubiquitin-proteasome pathway in intact mammalian cells because, upon activation, they are rapidly ubiquitinated and degraded. Using mass spectrometry, we previously examined the ubiquitination of IP(3)R1 in αT3-1 pituitary gonadotrophs and found that IP(3)R1 ubiquitination is highly complex, with receptors being modified at multiple sites by monoubiquitin and polyubiquitin chains formed through both Lys-48 and Lys-63 linkages (Sliter, D.

SPFH1 and SPFH2 mediate the ubiquitination and degradation of inositol 1,4,5-trisphosphate receptors in muscarinic receptor-expressing HeLa cells.

Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum (ER) membrane calcium channels that, upon activation, become substrates for the ER-associated degradation (ERAD) pathway. While it is clear that IP(3) receptors are polyubiquitinated and are transferred to the proteasome by a p97-based complex, currently very little is known about the proteins that initially select activated IP(3) receptors for ERAD. Here, we have transfected HeLa cells to stably express m3 muscarinic receptors to allow for the study of IP(3) receptor ERAD in this cell type, and show that IP(3) receptors are polyubiquitinated and then degraded by the proteasome in response to carbachol, a muscarinic agonist.

When worlds collide: IP(3) receptors and the ERAD pathway.

While cell signaling devotees tend to think of the endoplasmic reticulum (ER) as a Ca(2+) store, those who study protein synthesis tend to see it more as site for protein maturation, or even degradation when proteins do not fold properly. These two worldviews collide when inositol 1,4,5-trisphosphate (IP(3)) receptors are activated, since in addition to acting as release channels for stored ER Ca(2+), IP(3) receptors are rapidly destroyed via the ER-associated degradation (ERAD) pathway, a ubiquitination- and proteasome-dependent mechanism that clears the ER of aberrant proteins. Here we review recent studies showing that activated IP(3) receptors are ubiquitinated in an unexpectedly complex manner, and that a novel complex composed of the ER membrane proteins SPFH1 and SPFH2 (erlin 1 and 2) binds to IP(3) receptors immediately after they are activated and mediates their ERAD.

Mass spectrometric analysis of type 1 inositol 1,4,5-trisphosphate receptor ubiquitination.

Inositol 1,4,5-trisphosphate (IP(3)) receptors form tetrameric channels in endoplasmic reticulum membranes of mammalian cells and mediate IP(3)-induced calcium mobilization. In response to various extracellular stimuli that persistently elevate IP(3) levels, IP(3) receptors are also ubiquitinated and then degraded by the proteasome. Here, for endogenous type 1 IP(3) receptor (IP(3)R1) activated by endogenous signaling pathways and processed by endogenous enzymes, we sought to determine the sites of ubiquitination and the composition of attached ubiquitin conjugates.