Biology subjects

Syska, C.

Publications and source records attributed to Syska, C..

3 recordsLinked to original sources

bioRxiv2025

Ist2 promotes lipid transfer by Osh6 via its membrane tethering and lipid scramblase activities

Lipid transfer proteins (LTPs) are required for the uneven distribution of lipids between cellular membranes, which is essential for many cell functions. In yeast, Osh6 is an LTP that exchanges phosphatidylserine (PS) with phosphatidylinositol 4-phosphate (PI(4)P) between the endoplasmic reticulum (ER) and the plasma membrane (PM), promoting the enrichment of PS in the PM. Here, we address why, to function optimally, Osh6 must bind to Ist2, an ER-resident lipid scramblase able to connect the ER to the PM via an intrinsically disordered region (IDR). We determined in vitro that Osh6 binds to the Ist2 IDR with micromolar affinity, whether empty or bound to its lipid ligands. Moreover, we found that Osh6 efficiently transfers PS at ER-PM contact sites if the Ist2 IDR has a minimal length and its binding site in the IDR is sufficiently removed from the ER surface. Next, we reconstituted the Osh6:Ist2 complex within artificial ER-PM contact sites and demonstrated that the association of Osh6 with Ist2 allows for a fast and directed PS flux between the connected membranes. We identified the Ist2 binding site on the Osh6 surface by validating structural models using our functional assays. Finally, we found that the Osh6-mediated PS transfer can be coupled to the PS scramblase activity of Ist2. These data unveil new functional partnerships between an LTP and a membrane tethering/scramblase protein and point to the general advantage of localizing these processes to membrane contact sites to ensure their efficiency.

biochemistry↗

bioRxiv2025

Ist2, a protein involved in phosphatidylserine transport, is an ER lipid scramblase

Lipid scramblases allow passive flip-flop of phospholipids between bilayer leaflets, thereby promoting membrane symmetry. At the endoplasmic reticulum (ER), where phospholipid synthesis is restricted to one of the two leaflets, scramblase activity should be essential for equilibrated membrane growth. However, phospholipid scramblases at the ER are poorly understood. The yeast protein Ist2 contains an ER domain and a cytosolic tail that binds the plasma membrane (PM) and participates in the transfer of phosphatidylserine (PS). Here, we show both in vitro and in silico that the ER- domain of Ist2, which bears homology to the TMEM16 proteins, possesses a lipid scramblase activity. Ist2 activity is not regulated by Ca2+, in contrast to TMEM16 proteins, but is affected by the lipid composition of the bilayer used in simulations. In cells, we do not find a strong impact of the scramblase domain of Ist2 in on PS distribution; however, its over-expression or deletion affects processes at the ER such as vesicular transport, lipid droplet biogenesis and general phospholipid transport, with a specific contribution of residues important for lipid scrambling. Our study therefore identifies the first dedicated phospholipid scramblase in yeast and demonstrates that membrane asymmetry can impact diverse membrane-remodeling processes at the ER.

cell biology↗

bioRxiv2024

N-acetyl-transferases required for iron uptake and aminoglycoside resistance promote virulence lipid production in M. marinum

Phagosomal lysis is a key aspect of mycobacterial infection of host macrophages. Acetylation is a protein modification mediated enzymatically by N-acetyltransferases (NATs) that impacts bacterial pathogenesis and physiology. To identify NATs required for lytic activity, we leveraged Mycobacterium marinum, a nontubercular pathogen and an established model for M. tuberculosis. M. marinum hemolysis is a proxy for phagolytic activity. We generated M. marinum strains with deletions in conserved NAT genes and screened for hemolytic activity. Several conserved lysine acetyltransferases (KATs) contributed to hemolysis. Hemolysis is mediated by the ESX-1 secretion system and by phthiocerol dimycocerosate (PDIM), a virulence lipid. For several strains, the hemolytic activity was restored by the addition of second copy of the ESX-1 locus. Using thin-layer chromatography (TLC), we found a single NAT required for PDIM and phenolic glycolipid (PGL) production. MbtK is a conserved KAT required for mycobactin siderophore synthesis and virulence. Mycobactin J exogenously complemented PDIM/PGL production in the {Delta}mbtK strain. The {Delta}mbtK M. marinum strain was attenuated in macrophage and Galleria mellonella infection models. Constitutive expression of either eis or papA5, which encode a KAT required for aminoglycoside resistance and a PDIM/PGL biosynthetic enzyme, rescued PDIM/PGL production and virulence of the {Delta}mbtK strain. Eis N-terminally acetylated PapA5 in vitro, supporting a mechanism for restored lipid production. Overall, our study establishes connections between the MbtK and Eis NATs, and between iron uptake and PDIM and PGL synthesis in M. marinum. Our findings underscore the multifunctional nature of mycobacterial NATs and their connection to key virulence pathways. Significance StatementAcetylation is a modification of protein N-termini, lysine residues, antibiotics and lipids. Many of the enzymes that promote acetylation belong to the GNAT family of proteins. M. marinum is a well-established as a model to understand how M. tuberculosis causes tuberculosis. In this study we sought to identify conserved GNAT proteins required for early stages of mycobacterial infection. Using M. marinum, we determined that several GNAT proteins are required for the lytic activity of M. marinum. We uncovered previously unknown connections between acetyl-transferases required for iron uptake and antimicrobial resistance, and the production of the unique mycobacterial lipids, PDIM and PGLOur data support that acetyl-transferases from the GNAT family are interconnected, and have activities beyond those previously reported.

microbiology↗