Search

Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a novel type of protein prenyltransferase, which we named geranylgeranyltransferase type‐III (GGTase‐III). GGTase‐III consists of prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the β subunit of RabGGTase. Using a biotinylated geranylgeranyl analogue, we identified the Golgi SNARE protein Ykt6 as a substrate of GGTase‐III. GGTase‐III transfers a geranylgeranyl group to mono‐farnesylated Ykt6, generating doubly prenylated Ykt6. The crystal structure of GGTase‐III in complex with Ykt6 provides structural basis for Ykt6 double prenylation. In GGTase‐III‐deficient cells, Ykt6 remained in a singly prenylated form, and the Golgi SNARE complex assembly was severely impaired. Consequently, the Golgi apparatus was structurally disorganized, and intra‐Golgi protein trafficking was delayed. Our findings reveal a fourth type of protein prenyltransferase that generates geranylgeranyl‐farnesyl Ykt6. Double prenylation of Ykt6 is essential for the structural and functional organization of the Golgi apparatus. Synopsis: Three types of protein prenyltransferases are known to catalyse protein farnesylation and geranylgeranylation in eukaryotic cells. Here, a fourth class formed by PTAR1 heterodimerization with RabGGTβ is found to drive double prenylation of Golgi SNARE Ykt6, which is critical for Golgi complex maintenance and function in mammalian cells. Human PTAR1 and RabGGTβ form a fourth type of protein prenyltransferase, named GGTase‐III.GGTase‐III transfers a geranylgeranyl group to mono‐farnesylated Ykt6, generating doubly‐prenylated Ykt6.Structural analyses reveal the catalytic mechanism of Ykt6 double prenylation.Doubly‐prenylated Ykt6 is required for maintaining structure and function of the Golgi apparatus. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 3,5-bisphosphate ( PtdIns(3,5) P2) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5) P2 using freeze-fracture electron microscopy. GST-ATG18-4× FLAG was used to label PtdIns(3,5) P2 and its binding to phosphatidylinositol 3-phosphate ( PtdIns(3)P) was blocked by an excess of the p40phox PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5) P2 was concentrated in intramembrane particle ( IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5) P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5) P2 metabolism is defective. In HeLa cells, PtdIns(3,5) P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5) P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5) P2-rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. [ABSTRACT FROM AUTHOR]

Highlights: [•] Blockade of ER-to-Golgi transport increases ApoB-crescents. [•] Brefeldin A, a cellular ADP-ribosylation activator, induces ApoB-crescents. [•] Different ADP-ribosylation inhibitors all suppress Brefeldin A-induced ApoB-crescent formation. [Copyright &y& Elsevier]

PGAM5, a mitochondrial protein phosphatase that is genetically and biochemically linked to PINK1, facilitates mitochondrial division by dephosphorylating the mitochondrial fission factor Drp1. At the onset of mitophagy, PGAM5 is cleaved by PARL, a rhomboid protease that degrades PINK1 in healthy cells, and the cleaved form facilitates the engulfment of damaged mitochondria by autophagosomes by dephosphorylating the mitophagy receptor FUNDC1. Here, we show that the function and localization of PGAM5 are regulated by syntaxin 17 (Stx17), a mitochondria‐associated membrane/mitochondria protein implicated in mitochondrial dynamics in fed cells and autophagy in starved cells. In healthy cells, loss of Stx17 causes PGAM5 aggregation within mitochondria and thereby failure of the dephosphorylation of Drp1, leading to mitochondrial elongation. In Parkin‐mediated mitophagy, Stx17 is prerequisite for PGAM5 to interact with FUNDC1. Our results reveal that the Stx17‐PGAM5 axis plays pivotal roles in mitochondrial division and PINK1/Parkin‐mediated mitophagy. Synopsis: Mitochondrial phosphatase PGAM5 has been shown to regulate mitophagy by dephosphorylating the mitophagy receptor FUNDC1. New data show that syntaxin 17 associates with PGAM5 and regulates its localization and Drp1 and FUNDC1 activation during normal and mitophagic division, respectively. Syntaxin 17 binds PGAM5 and regulates its localization and activity.Syntaxin 17 promotes Drp1 activity by facilitating its PGAM5‐mediated dephosphorylation.Syntaxin 17 is required for the association of PGAM5 with the mitophagy receptor FUNDC1 in mitophagy.Syntaxin 17 and PGAM5 maintain mitochondrial architecture in Drosophila. Syntaxin 17 supports PGAM5‐mediated dephosphorylation and activation of fission factor Drp1 in healthy cells by regulating PGAM5 localization. [ABSTRACT FROM AUTHOR]