Structural and mechanistic insights into phospholipid transfer by Ups1–Mdm35 in mitochondria

Abstract

Eukaryotic cells are compartmentalized into membrane-bounded organelles whose functions rely on lipid trafficking to achieve membrane-specific compositions of lipids. Here we focused on the Ups1–Mdm35 system, which mediates phosphatidic acid (PA) transfer between the outer and inner mitochondrial membranes, and determined the X-ray structures of Mdm35 and Ups1–Mdm35 with and without PA. The Ups1–Mdm35 complex constitutes a single domain that has a deep pocket and flexible Ω-loop lid. Structure-based mutational analyses revealed that a basic residue at the pocket bottom and the Ω-loop lid are important for PA extraction from the membrane following Ups1 binding. Ups1 binding to the membrane is enhanced by the dissociation of Mdm35. We also show that basic residues around the pocket entrance are important for Ups1 binding to the membrane and PA extraction. These results provide a structural basis for understanding the mechanism of PA transfer between mitochondrial membranes.

Advances on the Transfer of Lipids by Lipid Transfer Proteins

“…Among the distinct movements of the LTP, transfer from Dn → Ac is only one; others will be Dn → Dn, Ac → Ac, and Ac → Dn. Assuming that the LTP interacts equally with Dn and Ac, the net transfers registered by gain of fluorescence (Dn → Ac) represent approximately (17% × 83%) = 14% of all transfers.(ii) Initial rate (the signal increase from t  = 5 − 15 s, after subtraction of background without LTP, as proportion of maximum signal) = (from Figure 5B in Watanabe 1.4%/s et al [21]). …”

“…Unlike intracellular TULIP proteins, PRELI/Ups proteins had been previously implicated in lipid metabolism [18]. Their predicted role as LTPs [19] has since been strengthened by PRELI/Ups crystal structures 20, 21, 22. These show PRELI/Ups in complex with a small accessory subunit TP53-regulated inhibitor of apoptosis-1, (TRIAP1; Mdm35 in yeast), which adds extra helices on to the StARkin β‐grip domain, structurally similar to an extra helix found in PI/phosphatidylcholine (PC) transfer protein (PITP) members of the StARkin superfamily (Table 1).…”

“…See Box 2 for potential sources of error. Abbreviations: Ac, acceptor; Dn, donor; PRELI, proteins of relevant evolutionary and lymphoid interest (in yeast called Ups for ‘ u n p roce s sed Mgm1’); TRIAP1, TP53-regulated inhibitor of apoptosis-1 (Mdm35 in yeast).

Detailed Analysis of PA Import into Mitochondria by Ups1 in Yeast (A) Evaluation of In Vitro PA Transfer Assays by Ups1/Mdm35 (Yeast Homologs of PRELI/TRIAP1) in Watanabe et al [21].Analysis of three factors yields a rate of 1/s for the in vitro transfer of PA by Ups1/Mdm35 complexesthat is shown in diagrammatic form in Figure 4A: (i) the proportion of transfers measured by fluorescence of NBD–PA, (ii) the rate of signal increase, and (iii) the number of NBD–PA transfers at equilibrium.(i) Donor (Dn) and acceptor (Ac) liposomes (ratio 1:4, both 100 nm diameter) have initial composition differences: Ac contains unlabelled PA (∼1250 nM in external leaflet). The PA in Dn is replaced by fluorescent NBD–PA (∼250 nM in external leaflet), which is quenched by rhodamine–PE present only in Dn.

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Mitochondrial Proteins Containing Coiled-Coil-Helix-Coiled-Coil-Helix (CHCH) Domains in Health and Disease

“…Within the IMS, Mdm35 stably interacts with members of the 184 evolutionary-conserved UPS/PRELI-like proteins UPS1 and UPS2 [63][64][65][66][67][68], which mediate 185 the transfer of phospholipids between the outer and inner mitochondrial membranes. For 186 instance, UPS1 controls the transfer of phosphatidic acid (PA) from the outer to the inner 187 membrane, where PA is channeled into a chain of enzymatic reactions resulting in the 188 production of cardiolipin (CL) [65,66,69].…”

Interaction between repressor Opi1p and ER membrane protein Scs2p facilitates transit of phosphatidic acid from the ER to mitochondria and is essential for INO1 gene expression in the presence of choline

“…1), primarily via the ERMES (35)(36)(37)(38). From the outer mitochondrial membrane, PA must be transferred to the inner mitochondrial membrane by a process requiring the Ups1p-Mdm35 complex (36,53,56). Once transferred to the inner mitochondrial membrane, PA serves as precursor to CDP-DAG, catalyzed by the mitochondrial CDP-DAG synthase, Tam41p ( Fig.…”