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Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipinenriched microdomains in which electron transport complexes are optimally assembled. Likely through the prohibitin functional interactome, SLP-2 then regulates mitochondrial biogenesis and function.

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Abstract. To study the role of (pro)collagen synthesis in the differentiation of rat L6 skeletal myoblasts, a specific inhibitor of collagen synthesis, ethyl-3,4-dihydroxybenzoate (DHB), was utilized. It is shown that DHB reversibly inhibits both morphological and biochemical differentiation of myoblasts, if it is added to the culture medium before the cell alignment stage. The inhibition is alleviated partially by ascorbate, which along with ot-ketoglutarate serves as cofactor for the enzyme, prolyl hydroxylase. DHB drastically decreases the secretion of procollagen despite an increase in the levels of the mRNA for pmod(I) and proot2(I) chains. Probably, the procollagen chains produced in the presence of DHB, being underhydroxylated, are unable to fold into triple helices and are consequently degraded in situ. Along with the inhibition of procollagen synthesis, DHB also decreases markedly the production of a collagen-binding glycoprotein (gp46) present in the ER. The results suggest that procollagen production and/or processing is needed as an early event in the differentiation pathway of myoblasts.

Transmembrane domains of receptor tyrosine kinases are increasingly seen as key modulatory elements in signaling pathways. The present work addresses problems surrounding expression, isolation, secondary structure recovery, and assembly into membranes, of the relatively large quantities of transmembrane peptides needed to investigate these pathways by NMR spectroscopy. We demonstrate significant correspondence between SDS-PAGE behavior of such peptides and their (2)H NMR spectra in lipid bilayer membranes. A 50-residue peptide, Neu(exp), containing the transmembrane portion of the receptor tyrosine kinase, Neu, was designed for expression in Escherichia coli. The sequence also contained 11-12 amino acids from each side of the transmembrane domain. The common problem of low expressivity of transmembrane peptides was encountered-likely associated with membrane toxicity of the desired gene product. This difficulty was overcome by expressing the peptide as a TrpE fusion protein in a pATH vector to target expression products to inclusion bodies, and subsequently removing the TrpE portion by cyanogen bromide cleavage. Inclusion bodies offered the additional benefits of reduced proteolytic degradation and simplified purification. The presence of a hexa-His tag allowed excellent recovery of the final peptide, while permitting use of denaturing solvents and avoiding the need for HPLC with its attendant adsorption losses. Isolated expressed peptides were found to be pure, but existed as high oligomers rich in beta-structure as evidenced by CD spectroscopy and SDS-PAGE behavior. Dissolution in certain acidic organic solvents led to material with increased alpha-helix content, which behaved in detergent as mixtures of predominantly monomers and dimers-a situation often considered to exist in cell membranes. For purposes of NMR spectroscopy, peptide alanine residues were deuterated in high yield during expression. The same acidic organic solvents used to dissolve and dissociate expressed transmembrane peptides proved invaluable for their assembly into lipid bilayers. Analogous transmembrane peptides from the human receptor tyrosine kinase, ErbB-2, demonstrated related phenomena.