Identification of the N-terminal transmembrane domain of StarD7 and its importance for mitochondrial outer membrane localization and phosphatidylcholine transfer

Abstract

StarD7 facilitates phosphatidylcholine (PC) transfer to mitochondria, and is essential for mitochondrial homeostasis. However, the molecular mechanism for PC transfer by protein remains poorly understood. Herein, we describe a putative novel transmembrane (TM) domain C-terminal to the mitochondria-targeting signal (MTS) sequence at the N-terminus of StarD7. The mature form of StarD7 is integrated and/or associated onto the outer leaflet of the outer mitochondrial membrane (OMM) in HEPA-1 and HepG2 cells. A truncated form of StarD7 lacking the TM domain is distributed in the inner space of the mitochondria, and cannot reverse mitochondrial abnormalities, such as complex formation and PC content, when re-expressed in StarD7-KO HEPA-1 cells. Re-expression of wild StarD7 can compensate these mitochondrial functions of StarD7-KO HEPA-1 cells. The precursor form of StarD7 is cleaved between Met76 and Ala77, and Ala77 and Ala78 in the TM domain to produce the mature form. These results suggest that StarD7 is anchored onto the OMM through its N-terminal TM domain, and the C-terminal START domain may extend into the cytoplasm and shuttle PC between the ER and OMM at the ER-mitochondria contact sites.

PARL partitions the lipid transfer protein STARD7 between the cytosol and mitochondria

“…To define how PARL affects sorting of STARD7, we first determined the localization of STARD7 within mitochondria and fractionated mitochondria purified from PARL +/+ cells by osmotic swelling (Fig A). In contrast to previous reports (Horibata et al , ), STARD7 was protected against externally added protease in intact mitochondria but was degraded upon disruption of the OM (Fig A). It thus shows a similar behavior than the i ‐AAA protease YME1L, which is active in the IMS, whereas matrix‐localized proteins such as mtHSP70 became accessible to externally added protease only upon solubilization of the IM (Fig A).…”

“…In contrast to this study, STARD7 has recently been reported to be localized to the mitochondrial OM (Horibata et al , ). To assess whether accumulation of STARD7 at the mitochondrial surface is sufficient to preserve mitochondrial activities, we replaced the mitochondrial targeting sequence and transmembrane region of STARD7 by the membrane anchor of the OM proteins TOMM70 or AKAP1 (Fig EV6A) and expressed these STARD7 variants in STARD7 −/− cells.…”

“…STARD7 has been identified as a LTP for PC in the cytosol (Horibata & Sugimoto, ; Horibata et al , , ). Our results reveal that IMS‐localized STARD7 represents a novel intramitochondrial LTP and shuttles PC between both mitochondrial membranes.…”

“…The loss of STARD7 in mouse hepatoma cells impairs the accumulation of PC in mitochondrial membranes as well as mitochondrial ATP production and cristae morphogenesis (Horibata et al , ). STARD7 has been localized at the mitochondrial OM and in the cytosol and was suggested to mediate PC transfer from the cytosol to mitochondrial membranes (Horibata & Sugimoto, ; Horibata et al , ). However, it remains unclear how this function can be reconciled with PARL‐mediated maturation of STARD7 at the IM (Saita et al , ).…”

A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7

“…Loss of StarD7 causes a significant reduction in mitochondrial PC levels, impaired respiratory activity, overproduction of reactive oxygen species, and disrupted cristae structures (31,32). StarD7 resides in the outer mitochondrial membrane (OMM) and is believed to shuttle PC between ER and OMM at ER-mitochondria contact sites (40,64). As ceramide biosynthesis occurs in mitochondria-associated ER membranes (MAMs; (61,62)) and is upregulated in response to various stress stimuli (8,9), it is conceivable that the local concentration of ceramides at ER-mitochondria contact sites can rise to a level where it affects StarD7-mediated mitochondrial PC import.…”