Analysis of Yeast Lipids

Schneiter, Roger; Daum, Günther

doi:10.1385/1-59259-958-3:075

Cited by 39 publications

(42 citation statements)

References 5 publications

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“…The lipids in the organic phase were washed with 0.034% (w/v) MgCl 2 , a 4:1 (v/v) mixture of 2 N KCl/methanol, and subsequently with a mixture of methanol/water/chloroform (48:47:3, per volume). Two-dimensional thin layer chromatography was used to separate the individual phospholipid classes (94). Finally, phospholipids were stained with iodine vapor, scraped off, and quantified (95).…”

Section: Methodsmentioning

confidence: 99%

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Schuler¹,

Bartolomeo

Mårtensson

et al. 2016

Journal of Biological Chemistry

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Two protein translocases transport precursor proteins into or across the inner mitochondrial membrane. The presequence translocase (TIM23 complex) sorts precursor proteins with a cleavable presequence either into the matrix or into the inner membrane. The carrier translocase (TIM22 complex) inserts multispanning proteins into the inner membrane. Both protein import pathways depend on the presence of a membrane potential, which is generated by the activity of the respiratory chain. The non-bilayer-forming phospholipids cardiolipin and phosphatidylethanolamine are required for the activity of the respiratory chain and therefore to maintain the membrane potential for protein import. Depletion of cardiolipin further affects the stability of the TIM23 complex. The role of bilayer-forming phospholipids like phosphatidylcholine (PC) in protein transport into the inner membrane and the matrix is unknown. Here, we report that import of presequence-containing precursors and carrier proteins is impaired in PC-deficient mitochondria. Surprisingly, depletion of PC does not affect stability and activity of respiratory supercomplexes, and the membrane potential is maintained. Instead, the dynamic TIM23 complex is destabilized when the PC levels are reduced, whereas the TIM22 complex remains intact. Our analysis further revealed that initial precursor binding to the TIM23 complex is impaired in PC-deficient mitochondria. We conclude that reduced PC levels differentially affect the TIM22 and TIM23 complexes in mitochondrial protein transport.Mitochondria fulfill essential functions for the survival of the cell like energy conversion to produce ATP, synthesis of amino acids, lipids, and heme, as well as the generation of iron-sulfur clusters. They contain about 1000 proteins in yeast and 1500 proteins in humans (1, 2). More than 99% of the mitochondrial proteins are synthesized as precursors on cytosolic ribosomes. Mitochondria contain a sophisticated system of protein translocases to import precursor proteins (3-9). The translocase of the outer membrane (TOM 3 complex) forms the general entry gate for most precursor proteins. After passage of the TOM channel, distinct protein translocases sort the preproteins into the different subcompartments: the outer and inner membrane as well as the two aqueous compartments, the matrix and intermembrane space.The majority of mitochondrial proteins are sorted into the inner membrane and the matrix. Two inner membrane-bound protein complexes mediate protein import. The presequence translocase (also termed TIM23 complex) transports precursor proteins with a cleavable presequence into the inner membrane and the matrix, whereas the carrier translocase (also termed TIM22 complex) inserts proteins with multiple transmembrane segments into the inner membrane (3-9). The membrane potential across the inner membrane provides the driving force for both protein import pathways and is generated by the activity of the respiratory chain. Presequence-containing preproteins are directly transferred from the ...

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Section: Methodsmentioning

confidence: 99%

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Schuler¹,

Bartolomeo

Mårtensson

et al. 2016

Journal of Biological Chemistry

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“…Subsequently, the organic phase was washed with 0.034% (w/v) MgCl 2 , a 4:1 (v/v) mixture of 2 N KCl/methanol and a mixture of methanol/water/chloroform (48:47:3, per vol.). Individual phospholipids were separated by two-dimensional thin-layer chromatography (83). For detection phospholipids were stained with iodine vapor, scrapped off, and quantified (84).…”

Section: Methodsmentioning

confidence: 99%

Phosphatidylcholine Affects the Role of the Sorting and Assembly Machinery in the Biogenesis of Mitochondrial β-Barrel Proteins

Schuler

Bartolomeo

Böttinger

et al. 2015

Journal of Biological Chemistry

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“…System B (whole-plate migration) consisted of petroleum ether-diethyl ether at 49:1 (vol/vol). Lipid classes were visualized by the MnCl 2 charring method (22). The silica plate was incubated for 1 min in a solution containing 120 ml of methanol, 120 ml of water, 0.8 g of MnCl 2 , and 8 ml of sulfuric acid and then heated in an oven at 100°C until the appearance of dark lipid spots.…”

Section: Methodsmentioning

confidence: 99%

Comparative Proteomic Analysis of Streptomyces lividans Wild-Type andppkMutant Strains Reveals the Importance of Storage Lipids for Antibiotic Biosynthesis

Maréchal

Decottignies

Marchand

et al. 2013

Appl Environ Microbiol

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dStreptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces.

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Analysis of Yeast Lipids

Cited by 39 publications

References 5 publications

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Phosphatidylcholine Affects the Role of the Sorting and Assembly Machinery in the Biogenesis of Mitochondrial β-Barrel Proteins

Comparative Proteomic Analysis of Streptomyces lividans Wild-Type andppkMutant Strains Reveals the Importance of Storage Lipids for Antibiotic Biosynthesis

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