Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and cellular functions. We now report that ablation of PITP␣ function leads to aponecrotic spinocerebellar disease, hypoglycemia, and intestinal and hepatic steatosis in mice. The data indicate that hypoglycemia is in part associated with reduced proglucagon gene expression and glycogenolysis that result from pancreatic islet cell defects. The intestinal and hepatic steatosis results from the intracellular accumulation of neutral lipid and free fatty acid mass in these organs and suggests defective trafficking of triglycerides and diacylglycerols from the endoplasmic reticulum. We propose that deranged intestinal and hepatic lipid metabolism and defective proglucagon gene expression contribute to hypoglycemia in PITP␣ ؊/؊ mice, and that hypoglycemia is a significant contributing factor in the onset of spinocerebellar disease. Taken together, the data suggest an unanticipated role for PITP␣ in with glucose homeostasis and in mammalian endoplasmic reticulum functions that interface with transport of specific luminal lipid cargoes. PITPs1 mobilize PtdIns or PtdCho between membrane bilayers in vitro (1,2). In vivo studies demonstrate that PITPs control the interface between membrane trafficking and lipid metabolic pathways in yeast (3-6). By contrast, the physiological functions for mammalian PITPs, which are structurally unrelated to yeast PITPs (7, 8), are not understood at either the cellular or organismal levels.Mammals express at least three soluble PITPs: PITP␣, PITP␤, and rdgB␤ (9 -11). PITP␣ and PITP␤ share 77% primary sequence identity, are encoded by distinct genes, and exhibit biochemical differences. Yet both PITP␣ and PITP␤ (and even yeast PITPs) function as soluble factors that stimulate various reconstitutions of PIP-dependent functions in permeabilized mammalian cells. These functions include regulated and constitutive membrane trafficking and phospholipase Cdependent signaling through G-protein-coupled receptors (12)(13)(14). Given the lack of PITP specificity in these assays, it remains unclear how faithful such reconstitutions are in reporting physiological functions for mammalian PITPs. Genetic studies are providing initial clues regarding PITP function in metazoans. An inherited form of light-enhanced retinal degeneration in Drosophila results from inactivation of a membrane-bound PITP (15). In mice, reduction of PITP␣ to 18% of wild-type levels is the basis for the vibrator neurodegenerative disorder (16,26). Gene ablation approaches suggest PITP␤ plays an essential housekeeping function, whereas PITP␣ is nonessential for ES cell viability and is not a quantitatively significant factor in membrane trafficking, PIP metabolism, or growth factor signaling in ES cells (17).In this report, we describe the consequences associated with ablation of PITP␣ function in the mouse. We find that PITP␣, although dispensable for prenatal development, is required for neonatal survival. PITP␣ Ϫ/Ϫ neonates suffer from a...

One-tenth of cytochrome c (cyt c) remains bound to the inner mitochondrial membrane (IMM) at physiological ionic strength (I; i.e. , I approximately 150 mM), exhibiting decreased electron transport (ET) activity. We now show that this form of membrane-bound cyt c (MB-cyt c) can be obtained in vitro and that binding to membranes at low I generates an additional conformation with higher ET activity. This low I bound form of MB-cyt c (MBL-cyt c) exhibited intrinsic ET rates similar to those of electrostatically bound cyt c (EB-cyt c). The ET activity of IMM-bound MB-cyt c approached slowly that of MBL-cyt c or EB-cyt c, suggesting that MB-cyt c converts to MBL-cyt c while bound to IMM. When maintained at physiological I, both forms of MB-cyt c were released from the membrane, indicating that they convert to an EB-cyt c-like form. This process may be very dynamic in cellular mitochondria, as binding and release for both MB-cyt c forms increased considerably with temperature. I-Dependent binding of MB-cyt c does not require IMM, and it can be reproduced using large or small unilamellar vesicles (SUV). Using SUV-cyt c complexes, we characterized the secondary structure of MB-cyt c and MBL-cyt c by circular dichroism. Conformational analysis revealed that cyt c binding as MB-cyt c decreases its alpha-helical content (70-79%) and increases its beta-sheet up to 135%. The secondary structure of MBL-cyt c was similar to that of EB-cyt c and soluble cyt c, with a modest increase in beta-sheet. Taken together, our experiments suggest that physiological cyt c exists in soluble and membrane-bound conformations with similar ET activity, which may exchange very rapidly, and that soluble hydrophilic proteins can bind transiently to biomembranes.

We have encapsulated actin rilaments in the presence and absence of various actin-binding proteins into lipid vesicles. These vesicles are approximately the same size as animal cells and can be characterized by the same optical microscopic and mechanical techniques used to study cells. We demonstrate that the initially spherical vesicles can be forced into asymmetric, irregular shapes by polymerization of the actin that they contain. Deformation of the vesicles requires that the actin filaments be on average at least =0.5 ,um long as shown by the effects of gelsolin, an actin rilament-nucleating protein. Filamin, a filament-crosslinking protein, caused the surfaces of the vesicles to have a smoother appearance. Heterogeneous distribution of actin filaments within the vesicles is caused by interfilament interactions and modulated by gelsolin and ifiamin. The vesicles provide a model system to study control ofcell shape and cytoskeletal organization, membranecytoskeleton interactions, and cytomechanics.The shapes and mechanical properties of animal cells are governed mainly by systems of cytoplasmic filaments collectively termed the cytoskeleton (1, 2). Ofthese systems, the actin microfilament system is the principal determinant of cellular viscoelastic properties (B.S. and E.L.E., in preparation) and is most directly involved in driving mechanical processes such as locomotion, cytokinesis, and phagocytosis (2, 3). As cells perform these functions, the organization of the actin cytoskeleton changes, probably under the control of actin-binding proteins that regulate the length and extent of crosslinking of the filaments (3-5). A model system in which cytoskeletal components could be reconstituted inside vesicles comparable in size to cells would be useful for studies of the regulation of cytoskeletal organization and the determination of cellular mechanical properties by the cytoskeleton.We have developed a model system of this kind for the actin filament system. Actin and actin-binding proteins have been encapsulated in lipid vesicles large enough (up to 20 jim in diameter) to be characterized by optical microscopy. The vesicles are large enough to be studied with biophysical techniques that measure, for example, actin diffusion by fluorescence photobleaching recovery (FPR) (6) or mechanical properties (1) ofindividual vesicles. The reconstitution of actin filaments with actin-binding proteins allows study of the effects of the latter on filament organization and distribution and the ability of the actin gel to drive morphological changes. In this work we have investigated the effects of gelsolin, which restricts the length of actin filaments, and of filamin, which crosslinks actin filaments (5). A striking observation is that vesicles are deformed when the actin inside them polymerizes. The extent of the deformation depends on the lengths of the resulting filaments. This observation provides experimental evidence for the speculation that actin filament polymerization can drive lamellar extension during ce...

We have shown that cytochrome c (cyt c) diffuses primarily in three dimensions in the intermembrane space (IMS) of intact mitochondria at physiological ionic strength (I). Recently, we found that a small percentage (11.2 +/- 2.1%) of endogenous cyt c remains bound to inner mitochondrial membranes (IMM) at high, physiological I (I = 150 mM), even after extensive washing with solutions at physiological I, overnight dialysis, changes in medium osmolarity, or further purification of IMM at high I using self-generating Percoll gradients. Measurements of heme c/heme a ratios, and electron transport (ET) reactions in which cyt c participates, confirmed the presence of a low amount of this I-resistant, membrane-bound form of cyt c (MB-cyt c), that had one third of the ET activity of electrostatically-bound cyt c (EB-cyt c), and which could not account for maximal ET rates. The amount of MB-cyt c was significantly increased above endogenous MB-cyt c by exposing KCl-washed IMM to increasing concentrations of exogenous cyt c. Also, subjecting large unilamellar vesicles (LUV) to successive cycles of cyt c binding/high I KCl-washes gave progressive increases in MB-cyt c. These protocols allowed in vitro characterization of MB-cyt c. The I at which binding takes place affects the affinity of cyt c for membranes, and oxidized cyt c had a greater intrinsic affinity for IMM or SUV than reduced cyt c. MB-cyt c appears to be bound partially by hydrophobic interactions since MB-cyt c was detected on negatively charged (asolectin) LUV and also on neutral, zwitterionic (phosphatidylcholine) LUV at high I. Consistent with the concentration-dependent changes in MB-cyt c, decreasing the IMS-volume of intact mitochondria (i.e., increasing th endogenous IMS-cyt c concentration) by metabolic or osmotic means increased the amount of MB-cyt c. After cyt c was delivered into the IMS by liposome-mediated low pH-induced fusion, resonance energy transfer showed a time-dependent cyt c-membrane proximity which was consistent with slow exchange of soluble IMS-entrapped cyt c molecules with a population bound to membranes at I = 150 mM. We conclude that, even though the majority of functional IMS-cyt c diffuses in three dimensions, a small portion remains firmly bound on the surface of the IMM under I conditions that are physiological for intact mitochondria. The occurrence of MB-cyt c may reflect an intrinsic conformational flexibility in cyt c, that allows a degree of membrane penetration and the formation of hydrophobic interactions which stabilize the membrane-bound form. The persistence of cyt c-membrane interactions under physiological I conditions indicates that cyt c-mediated ET in the IMS involves both fast (3D-diffusion) and slow (2D-diffusion) pathways for electron transfer.

Abstract. The electrostatic interactions of cytochrome c with its redox partners and membrane lipids, as well as other protein interactions and biochemical reactions, may be modulated by the ionic strength of the intermembrane space of the mitochondrion. FITC-BSA was used to determine the relative value of the mitochondrial intermembrane ionic strength with respect to bulk medium external to the mitochondrial outer membrane. FITC-BSA exhibited an ionic strength-dependent fluorescence change with an affinity in the mM range as opposed to its pH sensitivity in the #M range. A controlled, low pH-induced membrane fusion procedure was developed to transfer FITC-BSA encapsulated in asolectin liposomes, to the intermembrane space of intact mitochondria. The fusion procedure did not significantly affect mitochondrial ultrastructure, electron transport, or respiratory control ratios. The extent of fusion of liposomes with the mitochondrial outer membrane was monitored by fluorescence dequenching assays using a membrane fluorescent probe (octadecylrhodamine B) and the soluble FITC-BSA fluorescent probe, which report membrane and contents mixing, respectively. Assays were consistent with a rapid, low pH-induced vesicle-outer membrane fusion and delivery of FITC-BSA into the intermembrane space. Similar affinities for the ionic strength-dependent change in fluorescence were found for bulk medium, soluble (9.8 ± 0.8 mM) and intermembrane spaceentrapped FITC-BSA (10.2 + 0.6 mM). FITC-BSA consistently reported an ionic strength in the intermembrane space of the functionally and structurally intact mitochondria within ±20% of the external bulk solution. These findings reveal that the intermembrane ionic strength changes as does the external ionic strength and suggest that cytochrome c interactions, as well as other protein interactions and biochemical reactions, proceed in the intermembrane space of mitochondria in the intact cell at physiological ionic strength, i.e., 100-150 mM.