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Alteration in mitochondrial fusion may regulate mitochondrial metabolism. Since the phospholipid cardiolipin (CL) is required for function of the mitochondrial respiratory chain, we examined the dynamics of CL synthesis in growing Hela cells immediately after and 12h post-fusion. Cells were transiently transfected with Mfn-2, to promote fusion, or Mfn-2 expressing an inactive GTPase for 24h and de novo CL biosynthesis was examined immediately after or 12h post-fusion. Western blot analysis confirmed elevated Mfn-2 expression and electron microscopic analysis revealed that Hela cell mitochondrial structure was normal immediately after and 12h post-fusion. Cells expressing Mfn-2 exhibited reduced CL de novo biosynthesis from [1,3-(3)H]glycerol immediately after fusion and this was due to a decrease in phosphatidylglycerol phosphate synthase (PGPS) activity and its mRNA expression. In contrast, 12h post-mitochondrial fusion cells expressing Mfn-2 exhibited increased CL de novo biosynthesis from [1,3-(3)H]glycerol and this was due to an increase in PGPS activity and its mRNA expression. Cells expressing Mfn-2 with an inactive GTPase activity did not exhibit alterations in CL de novo biosynthesis immediately after or 12h post-fusion. The Mfn-2 mediated alterations in CL de novo biosynthesis were not accompanied by alterations in CL or monolysoCL mass. [1-(14)C]Oleate incorporation into CL was elevated at 12h post-fusion indicating increased CL resynthesis. The reason for the increased CL resynthesis was an increased mRNA expression of tafazzin, a mitochondrial CL resynthesis enzyme. Ceramide-induced expression of PGPS in Hela cells or in CHO cells did not alter expression of Mfn-2 indicating that Mfn-2 expression is independent of altered CL synthesis mediated by elevated PGPS. In addition, Mfn-2 expression was not altered in Hela cells expressing phospholipid scramblase-3 or a disrupted scramblase indicating that proper CL localization within mitochondria is not essential for Mfn-2 expression. The results suggest that immediately post-mitochondrial fusion CL de novo biosynthesis is "slowed down" and then 12h post-fusion it is "upregulated". The implications of this are discussed., (Copyright 2010 Elsevier B.V. All rights reserved.)

The first enzymatic step in the biosynthesis of steroid hormones occurs in the mitochondrial inner membrane and is dependent on the mobilization of cholesterol from cellular stores. We report on the isolation of a human cDNA which encodes a mitochondrial protein called steroidogenic acute regulatory (StAR) protein, implicated in transport of cholesterol into mitochondria. Nucleotide and predicted amino acid sequence analyses indicate that the human and murine polypeptides are highly conserved, sharing 87% identity with an overall homology of 92%. Analysis of the distribution of StAR mRNA transcripts in human tissues by Northern blotting reveals several mRNA species, the most abundant of which is a 1.8 kb mRNA transcript present in testes, ovaries and kidneys. Using in vitro translated protein, we demonstrate that the StAR gene product can be efficiently imported into exogenously added mitochondria.

Mitochondria with a ruptured outer membrane exhibited impaired import into this membrane of an outer membrane fusion protein containing the signal-anchor sequence of Mas70p. However, the Mas70p signal-anchor efficiently targeted and inserted the protein directly into exposed regions of the inner membrane. Import into the inner membrane was dependent on delta psi and this dependence was due to the presence of the positively-charged amino acids located at positions 2, 7, and 9 of the signal-anchor. In contrast to wild-type signal-anchor, mutants lacking the positively-charged residues mediated import into the inner membrane in both the presence and absence of delta psi. The results suggest two conclusions: (1) delta psi-dependent import of the signal-anchor sequence was due exclusively to an effect of delta psi on the positively-charged domain of the signal-anchor, rather than to an effect of delta psi on a property of the inner membrane import machinery; (2) in the absence of delta psi, the positively-charged domain of the signal-anchor prevented the otherwise import-competent signal-anchor from inserting into the membrane. This suggests that the positively-charged domain leads import across the inner membrane, and that delta psi is required to vectorially clear this domain in order to allow the distal region of the signal-anchor to enter the translocation pathway. The implications of these findings on the mechanism of import into the mitochondrial inner membrane and matrix are discussed.