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Glutathione transferase P1 (GSTP1) is a multi-functional protein that protects cells from electrophiles by catalyzing their conjugation with glutathione, and contributes to the regulation of cell proliferation, apoptosis, and signalling. GSTP1, usually described as a cytosolic enzyme, can localize to other cell compartments and we have reported its strong association with the plasma membrane. In the current study, the hypothesis that GSTP1 is palmitoylated and this modification facilitates its dynamic localization and function was investigated. Palmitoylation is the reversible post-translational addition of a 16-C saturated fatty acid to proteins, most commonly on Cys residues through a thioester bond. GSTP1 in MCF7 cells was modified by palmitate, however, GSTP1 Cys to Ser mutants (individual and Cys-less) retained palmitoylation. Treatment of palmitoylated GSTP1 with 0.1 N NaOH, which cleaves ester bonds, did not remove palmitate. Purified GSTP1 was spontaneously palmitoylated in vitro and peptide sequencing revealed that Cys48 and Cys102 undergo S- palmitoylation, while Lys103 undergoes the rare N- palmitoylation. N -palmitoylation occurs via a stable NaOH-resistant amide bond. Analysis of subcellular fractions of MCF7-GSTP1 cells and a modified proximity ligation assay revealed that palmitoylated GSTP1 was present not only in the membrane fraction but also in the cytosol. GSTP1 isolated from E . coli , and MCF7 cells (grown under fatty acid free or regular conditions), associated with plasma membrane-enriched fractions and this association was not altered by palmitoyl CoA. Overall, GSTP1 is modified by palmitate, at multiple sites, including at least one non-Cys residue. These modifications could contribute to regulating the diverse functions of GSTP1.

The mitochondria‐associated membrane (MAM) is a domain of the endoplasmic reticulum (ER) that mediates the exchange of ions, lipids and metabolites between the ER and mitochondria. ER chaperones and oxidoreductases are critical components of the MAM. However, the localization motifs and mechanisms for most MAM proteins have remained elusive. Using two highly related ER oxidoreductases as a model system, we now show that palmitoylation enriches ER‐localized proteins on the MAM. We demonstrate that palmitoylation of cysteine residue(s) adjacent to the membrane‐spanning domain promotes MAM enrichment of the transmembrane thioredoxin family protein TMX. In addition to TMX, our results also show that calnexin shuttles between the rough ER and the MAM depending on its palmitoylation status. Mutation of the TMX and calnexin palmitoylation sites and chemical interference with palmitoylation disrupt their MAM enrichment. Since ER‐localized heme oxygenase‐1, but not cytosolic GRP75 require palmitoylation to reside on the MAM, our findings identify palmitoylation as key for MAM enrichment of ER membrane proteins. Mitochondria‐associated ER membranes (MAM) recently emerged as critical for cellular signal transduction and metabolism. This study identifies palmitoylation as novel mechanism for targeted localization of ER‐transmembrane proteins to MAM domains.

Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, is critical for membrane targeting and cell signaling. Because cancer cells often have increased N-myristoyltransferase (NMT) expression, NMTs were proposed as anti-cancer targets. To systematically investigate this, we performed robotic cancer cell line screens and discovered a marked sensitivity of hematological cancer cell lines, including B-cell lymphomas, to the potent pan-NMT inhibitor PCLX-001. PCLX-001 treatment impacts the global myristoylation of lymphoma cell proteins and inhibits early B-cell receptor (BCR) signaling events critical for survival. In addition to abrogating myristoylation of Src family kinases, PCLX-001 also promotes their degradation and, unexpectedly, that of numerous non-myristoylated BCR effectors including c-Myc, NFκB and P-ERK, leading to cancer cell death in vitro and in xenograft models. Because some treated lymphoma patients experience relapse and die, targeting B-cell lymphomas with a NMT inhibitor potentially provides an additional much needed treatment option for lymphoma. N-myristoyltransferases (NMTs) target many signaling proteins to membranes. Here the authors show an NMT inhibitor named PCLX-001 selectively kills lymphoma cells by shutting down their main survival signaling pathway and offers an additional treatment strategy for lymphoma patients.

Background Rab32 is a small GTPase associated with multiple organelles but is particularly enriched at the endoplasmic reticulum (ER). Here, it controls targeting to mitochondria-ER contacts (MERCs), thus influencing composition of the mitochondria-associated membrane (MAM). Moreover, Rab32 regulates mitochondrial membrane dynamics via its effector dynamin-related protein 1 (Drp1). Rab32 has also been reported to induce autophagy, an essential pathway targeting intracellular components for their degradation. However, no autophagy-specific effectors have been identified for Rab32. Similarly, the identity of the intracellular membrane targeted by this small GTPase and the type of autophagy it induces are not known yet. Results To investigate the target of autophagic degradation mediated by Rab32, we tested a large panel of organellar proteins. We found that a subset of MERC proteins, including the thioredoxin-related transmembrane protein TMX1, are specifically targeted for degradation in a Rab32-dependent manner. We also identified the long isoform of reticulon-3 (RTN3L), a known ER-phagy receptor, as a Rab32 effector. Conclusions Rab32 promotes degradation of mitochondrial-proximal ER membranes through autophagy with the help of RTN3L. We propose to call this type of selective autophagy “MAM-phagy”.

Rab32 is a small GTPase associated with multiple organelles but is particularly enriched at the endoplasmic reticulum (ER). Here, it controls targeting to mitochondria-ER contacts (MERCs), thus influencing composition of the mitochondria-associated membrane (MAM). Moreover, Rab32 regulates mitochondrial membrane dynamics via its effector dynamin-related protein 1 (Drp1). Rab32 has also been reported to induce autophagy, an essential pathway targeting intracellular components for their degradation. However, no autophagy-specific effectors have been identified for Rab32. Similarly, the identity of the intracellular membrane targeted by this small GTPase and the type of autophagy it induces are not known yet. To investigate the target of autophagic degradation mediated by Rab32, we tested a large panel of organellar proteins. We found that a subset of MERC proteins, including the thioredoxin-related transmembrane protein TMX1, are specifically targeted for degradation in a Rab32-dependent manner. We also identified the long isoform of reticulon-3 (RTN3L), a known ER-phagy receptor, as a Rab32 effector. Rab32 promotes degradation of mitochondrial-proximal ER membranes through autophagy with the help of RTN3L. We propose to call this type of selective autophagy \"MAM-phagy\".

Rab32 is a small GTPase associated with multiple organelles but is particularly enriched at the endoplasmic reticulum (ER). Here, it controls targeting to mitochondria-ER contacts (MERCs), thus influencing composition of the mitochondria-associated membrane (MAM). Moreover, Rab32 regulates mitochondrial membrane dynamics via its effector dynamin-related protein 1 (Drp1). Rab32 has also been reported to induce autophagy, an essential pathway targeting intracellular components for their degradation. However, no autophagy-specific effectors have been identified for Rab32. Similarly, the identity of the intracellular membrane targeted by this small GTPase and the type of autophagy it induces are not known yet. To investigate the target of autophagic degradation mediated by Rab32, we tested a large panel of organellar proteins. We found that a subset of MERC proteins, including the thioredoxin-related transmembrane protein TMX1, are specifically targeted for degradation in a Rab32-dependent manner. We also identified the long isoform of reticulon-3 (RTN3L), a known ER-phagy receptor, as a Rab32 effector. Rab32 promotes degradation of mitochondrial-proximal ER membranes through autophagy with the help of RTN3L. We propose to call this type of selective autophagy \"MAM-phagy\".

Thioredoxin-related transmembrane proteins (TMX) of the endoplasmic reticulum (ER) have emerged as key regulators of ER membrane properties. Within the ER lumen, TMX proteins and other ER redox enzymes determine oxidative conditions, which control the formation of ER-mitochondria membrane contacts (ERMCS) and determine their function. ERMCS exhibit cytoplasmic redox nanodomains, derived from ER and mitochondrial reactive oxygen species (ROS), whose mechanistic regulation is uncharacterized. Our research has identified the ER protein TMX2, which uses its unique cytosolic thioredoxin domain to prevent cytosolic sulfenylation of mitochondrial outer membrane proteins such as TOM70 through a functional interaction with peroxiredoxin-1 (PRDX1). By doing so, TMX2 interferes with the TOM70 ERMCS tethering function and reduces mitochondrial Ca2+ flux and metabolism. Recently, TMX2 mutations have been identified to cause a neurodevelopmental disorder with microcephaly, cortical malformations, and spasticity (NEDMCMS). Using TMX2-mutated NEDMCMS patient cells, we demonstrate that compromising TMX2 through mutation reproduces mitochondrial defects. In a fly in vivo model, TMX2 knockdown manifests predominantly in glial cells. Our results therefore provide important mechanistic insight into NEDMCMS and mechanistically link TMX2-mediated control of ERMCS to brain development and function. The transmembrane thioredoxin-related TMX2 prevents TOM70 sulfenylation at ERMCS, thus maintaining normal mitochondria metabolism in wild-type cells. TMX2 knockout leads to TOM70 sulfenylation and tight ERMCS formation. This then increases ROS production, unbalances mitochondrial lipids, and relatively shifts OXPHOS electron supply to complex II.