Isolation of Organellar Membranes from S. cerevisiae
The aim of the present work was to determine qualitative differences between the lipid molecular species composition of distinct organellar membranes. To minimize alterations in the lipid composition due to culture variations, as many membranes as possible were isolated from a single batch of cells. The fractionation strategy designed to simultaneously isolate nine different organellar membranes is outlined in . A 10 liter fermentation culture of wild-type cells was grown to late exponential phase, harvested, and one fifth of the total cell mass was used for the isolation of the plasma membrane fraction, which required disintegration of intact cells by glass beads (Serrano 1988
). The remainder of the culture was converted to spheroplasts, split into three aliquots, each of which was then specifically processed for the isolation of particular fractions, i.e., nuclei (Hurt et al. 1988
; Aris and Blobel 1991
), vacuoles and lipid particles (Uchida et al. 1988
; Leber et al. 1994
), and mitochondria, which were further subfractionated into inner and outer mitochondrial membranes and contact sites (intermediate density fraction; Pon et al. 1989
). The postmitochondrial supernatant was subfractionated into heavy (40,000 g
) and light (100,000 g
) microsomes, and the soluble cytosolic fraction (Zinser and Daum 1995
). Since Golgi membranes could not be isolated in sufficient yield and quality from a late exponential phase culture, an early exponential phase culture of the same wild-type strain was processed in parallel for the isolation of this organelle (Lupashin et al. 1996
). The isolation of peroxisomes from yeast requires prior induction of the organelle by oleic acid. Peroxisomes were thus isolated from an independent culture cultivated under inducing conditions (Veenhuis et al. 1987
Outline of the fractionation scheme designed to simultaneously isolate nine subcellular membrane fractions from one single 10 l culture of S. cerevisiae wild-type cells.
Biochemical Characterization of Subcellular Fractions
Biochemical and morphological criteria were subsequently employed to assess the quality of the isolated membranes. Relative enrichment and degree of cross-contamination of the fractions was judged by immunoblot analysis with antibodies against the following marker proteins: Kar2p/BiP, a HSP70 family member and soluble lumenal ER protein, served as marker for the ER (Normington et al. 1989
); α1,2-mannosyltransferase (Kre2p; Lussier et al. 1995
), was used as marker for the Golgi membrane; Gas1p, a glycosylphosphatidylinositol (GPI)-anchored membrane protein (Conzelmann et al. 1988
), was the marker for the plasma membrane; CPY served as a lumenal marker for the vacuole; 3-phosphoglycerate kinase (PGK) was used as marker for soluble cytosolic protein; Δ24-sterol C-methyltransferase (Erg6p) served as marker protein for the lipid particle fraction (Leber et al. 1994
); Acyl-CoA oxidase (Pox1p) was a marker for peroxisomes (Thieringer et al. 1991
); porin was used as marker for the outer mitochondrial membrane (Daum et al. 1982
); ADP/ATP carrier protein (Aac1p) marked the inner mitochondrial membrane; and malate dehydrogenase (MDH) was used as marker of the mitochondrial matrix. Immunoblot analyses of all the subcellular fractions probed with these antibodies are shown in and a quantification of the results is given in .
Figure 2 Immunoblot analysis of subcellular fractions. 10 μg protein of the cell homogenate (10× Hom) and 1 μg of each of the subcellular fractions were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with antibodies (more ...)
Relative Enrichment of Marker Proteins in Subcellular Fractions
As shown in and , the plasma membrane was the subcellular fraction with the highest enrichment for its marker protein. The GPI-anchored Gas1p was enriched 185.5-fold. The isolated nuclei were 3.7-fold enriched for Kar2p. Enrichment factors of 10–15 for ER proteins in isolated yeast nuclear fractions have been reported for cells harvested at an early logarithmic phase (Hurt et al. 1988
; Aris and Blobel 1991
). The lower enrichment seen in our preparations likely is due to the late growth phase at which these cells were harvested. Kar2p was detected in various fractions, which is in line with the contamination of most organelles by the poorly defined microsomes.
The Golgi membrane was 23.5-fold enriched for the medial-Golgi apparatus marker Kre2p, which was also detected in the heavy microsome fraction (2.4-fold). Based on enzyme activity, this particular Golgi membrane fraction has been reported to be 205-fold enriched for the cis-Golgi membrane localized GDPase (Lupashin et al. 1996
). Since early and late Golgi compartments differ in density (Cunningham and Wickner 1989
), the Golgi fraction analyzed in this study is more representative of an early Golgi compartment when compared with the Kex2p enriched late Golgi compartment whose lipid composition has previously been analyzed (McGee et al. 1994
; Leber et al. 1995
The lipid particle fraction was highly enriched for its marker protein Erg6p, but also contained Kar2p, consistent with the proposed relationship of this compartment with the ER membrane (Leber et al. 1998
The vacuolar fraction was 15.2-fold enriched in the lumenal vacuolar CPY. Mitochondria were enriched for porin, a major protein of the outer mitochondrial membrane (Daum et al. 1982
), and for the ADP/ATP carrier protein, Aac1p, of the inner mitochondrial membrane. Both markers were further enriched in their corresponding subfractions. The marker protein for the mitochondrial matrix, MDH, was enriched in the mitochondrial fraction, but was also present in the inner mitochondrial membrane fraction, as has been observed previously (Zinser and Daum 1995
The dense microsomal fraction (40,000 g) was 7.0-fold enriched for Kar2p. None of the marker proteins were selectively enriched in the light microsomal fraction (100,000 g). The soluble cytosolic fraction was 5.2-fold enriched for phosphoglycerate kinase and did not show any enrichment for other marker proteins tested.
Peroxisomes were enriched ~30-fold for Pox1p relative to the corresponding homogenate (not shown), with no major cross-contaminations detectable.
Morphological Characterization of Membrane Fractions
To further assess the purity and homogeneity of the isolated fractions, membranes were fixed and examined by ultra-thin section EM. The results of this analysis are shown in . A distinct membrane/vesicle morphology for each of the isolated fractions was readily visible and the fractions appeared homogenous for the type of membrane isolated. The 40,000 g microsomal fraction contained the most morphologically inhomogeneous materials, such as big and small vesicles next to stacked sheets of membranes.
Figure 3 Morphological analysis of subcellular membrane fractions. Subcellular fractions were isolated as outlined in , fixed, and processed for EM as described in Materials and Methods. PM, plasma membrane; LP, lipid particles; Vac, vacuoles; (more ...)
Lipid bilayer thickness has been discussed as a possible sorting determinant for integral membrane proteins (Bretscher and Munro 1993
). We thus measured membrane thickness of the different fractions on high magnification prints of electron micrographs. The values obtained from this analysis are listed in . The average thickness of the membranes was 7.1 ± 0.4 nm. The plasma membrane was significantly thicker (9.2 ± 0.4 nm), and the lipid particle membrane was significantly thinner (4.5 ± 0.4 nm), as expected for a lipid monolayer that delineates the lipid particles (Leber et al. 1994
Thickness of Subcellular Membranes
Nano-ESI-MS/MS Analysis of the Lipid Composition of Subcellular Membranes
Lipids from the various subcellular membrane fractions were extracted, their phosphate content was determined, and set in relation to the protein content of the respective fraction (). The ergosterol content of the membranes was determined by quantitative nano-ESI-MS/MS analysis of lipid extracts containing a defined amount of [13
C]cholesterol as internal standard, added to the fractions before lipid extraction (Sandhoff et al. 1999
). Ratios of phospholipid to ergosterol content of the subcellular fractions are also listed in . These analyses complement previously published data (Zinser et al. 1991
, Zinser et al. 1993
), and show that the yeast plasma membrane has a phospholipid to ergosterol ratio of 2.2, which is comparable to a value of 1.9–2.9 reported for higher eukaryotic cells (Lange et al. 1989
; Allan and Kallen 1994
; van Meer 1998
). Limiting amount of material did not allow quantification of the phospholipid content of the Golgi, nuclei, and mitochondrial fractions, which are therefore not represented in .
Phospholipid and Sterol Composition of Subcellular Membranes
Lipid extracts of the subcellular membrane fractions were then analyzed by nano-ESI-MS/MS. A comparison of negative ion mode spectra of the different membranes is shown in . For peak assignment, each major ion was subjected to product ion scan analyses (data not shown). Interpretation of the spectra was facilitated by the relative simple fatty acid composition of S
phospholipids. As can be seen from the overview shown in , the plasma membrane differed most from all other fractions. Free ceramide (Cer-C, containing phytosphingosine with a α-hydroxylated C26 fatty acid) was readily detectable in this membrane fraction, whereas the mature sphingolipids (M(IP)2
C, IPC-C, and IPC-D) were also detected in the heavy microsome fraction (indicated in black in ). The m/z values of these sphingolipids were identical to those reported previously (Hechtberger et al. 1994
; for review see Lester and Dickson 1993
; Dickson 1998
; Schneiter 1999
Figure 4 Overview of the lipid molecular species distribution in different subcellular membranes. Negative ion mass spectra of the m/z range 500–1,000 of unprocessed lipid extracts prepared from the subcellular membranes indicated. Individual lipid classes (more ...)
To determine the relative abundance of each molecular species within its lipid class, scans specific for single phospholipid classes were recorded for every membrane fraction (data not shown; Brügger et al. 1997
). Specifically, the molecular species profile of PS was calculated from positive mode ion scans specific for the neutral loss of m/z 185 (specific for serine phosphate; and ). The molecular species profile of PE was calculated from positive mode ion scans specific for the neutral loss of m/z 141 (specific for ethanolamine phosphate; ). The molecular species profile of PC was calculated from positive ion precursor scans for m/z 184 (specific for choline phosphate; ). The molecular species profile of PI was calculated from negative ion precursor scans for m/z 241 (specific for the dehydration product of inositol phosphate; ).
PS Species of Yeast Subcellular Membranes
Plasma Membrane PS Species of Wild-type and elo3Δ Mutant Cells
PE Species of Yeast Subcellular Membranes
PC Species of Yeast Subcellular Membranes
PI Species of Yeast Subcellular Membranes
Since peroxisomes were isolated from cells cultivated in the presence of oleic acid (C18:1), the lipid molecular species profile of this membrane was shifted dramatically towards oleic acid-containing species (; , VI–VIII). For the comparison of the molecular species profiles of the different membranes that follow, peroxisomes were thus not further taken into account. Moreover, molecular species containing double desaturated acyl chains were observed in peroxisomal phospholipids (, VI–VIII). These are due to contaminations of the oleic acid preparation used to supplement the culture media by C18:2, as revealed by GC-MS analysis (data not shown).
A molecular species profile, calculated by averaging the values of ten different membranes, not including peroxisomes, is listed in and . In the right-most column of these Tables, the percentage of saturated acyl chains in the respective phospholipid class is given for each of the subcellular membranes.
Molecular Species of PS
Averaged over all membrane fractions, PS 34:1 (i.e., composed of oleic acid [C18:1] and palmitic acid [C16:0]) constituted the main species of PS with 40.9%. This was followed by PS 34:2 (i.e., composed of oleic acid [C18:1] and palmitoleic acid [C16:1]) with 30.6%, PS 32:1 (15.7%), and PS 32:2 (10.2%). In the plasma membrane, PS 34:1 was the most prominent species (64.1%). Surprisingly, this rise in PS 34:1 at the plasma membrane was compensated by a greatly diminished PS 32:2 level (1.9%). A similar, albeit less pronounced, reduction was observed for PS 34:2 (16.8%), suggesting that PS species containing two unsaturated fatty acids are replaced by and/or remodeled to species containing one saturated fatty acid at the plasma membrane.
Interestingly, in the lipid particle monolayer membrane, a PS profile contrasting that of the plasma membrane was observed. Diunsaturated fatty acid-substituted PS species, i.e., PS 34:2 (40.0%) and PS 32:2 (25.0%), were enriched at the expense of saturated fatty acid-containing species. The Golgi membrane, on the other hand, was greatly enriched in the short chain lipid PS 28:0, i.e., containing C14:0/C14:0 (10.6%).
Yeast has a plasma membrane phospholipid asymmetry typical of eukaryotic cells, with PS mainly, if not exclusively, in the inner leaflet, and sterols and sphingolipids in the outer leaflet (Devaux 1991
; Schroit and Zwaal 1991
; Cerbon and Calderon 1995
). The yeast plasma membrane has been estimated to contain most of the PS (90%), PE (80–90%), and PI (85%) in the inner leaflet and the sphingolipids (30% of total phospholipids) on the outer leaflet (Patton and Lester 1991
; Hechtberger et al. 1994
; Cerbon and Calderon 1995
; Balasubramanian and Gupta 1996
). The acyl chain composition of the sphingolipids in yeast is unusual in that they contain the saturated very long chain C26:0 fatty acid (Lester and Dickson 1993
; Hechtberger et al. 1994
; Schneiter and Kohlwein 1997
). The observation that monounsaturated PS species were greatly enriched at the expense of diunsaturated species suggested that the C26:0 containing sphingolipids may affect the acyl chain composition of lipids in the inner leaflet of the plasma membrane. To test this hypothesis, the PS profile of plasma membrane isolated from a yeast mutant, elo3
Δ (also known as SUR4/APA1/VBM1/SRE1/YLR372w
; David et al., 1988; García-Arranz et al. 1994
; Silve et al. 1996
; Oh et al. 1997
), that is defective in the final step of elongating the C24 to the C26 fatty acid, and hence, does not contain any C26 fatty acid, was analyzed. Compared with the corresponding wild-type strain, in the plasma membrane from the elo3
Δ mutant, PS 34:2 is the most abundant species (40.3%), followed by PS 32:2 (24.6%), PS 32:1 (15.3%), and PS 34:1 (8.2%; see ; ). This dramatic shift towards diunsaturated fatty acid-containing species in cells that synthesize C24-substituted sphingolipids thus suggests that the precise acyl chain length of very long chain fatty acid-substituted sphingolipids affects the acyl chain composition of neighboring glycerophospholipids.
Figure 5 PS profile of the plasma membrane in elo3Δ mutant cells. Positive ion mass spectra specific for molecular species of PS (neutral loss of m/z 185) in the m/z range 700–800 of unprocessed lipid extracts prepared from plasma membranes of (more ...)
Molecular Species of PE
Averaged over all membranes, the most abundant PE species was PE 34:2 (36.4%), followed by PE 32:2 (25.3%), PE 34:1 (15.5%), and PE 32:1 (14.8%). The tendency towards enrichment of saturated species (PE 32:1 and PE 34:1) at the expense of diunsaturated species (PE 32:2 and PE 34:2) at the plasma membrane, noted for PS, was also observed for PE (see ). Lipid particles were again enriched in the diunsaturated PE species, PE 32:2, as was the case for PS.
Molecular Species of PC
The molecular species profile of PC did not display significant variation between the different membranes (). Overall, PC 32:2 (40.0%) was the most abundant species, followed by the 34:2 (25.4%), 32:1 (14.9%), and 34:1 (9.3%) species.
Molecular Species of PI
The molecular species profile of PI was generally more heterogeneous than that of the other glycerophospholipids. This was mainly due to the fact that disaturated species of PI, but not of PS, PE, or PC, were present in all of the membranes (see ). The most abundant PI species was PI 34:1 (34.0%), followed by PI 32:1 (21.8%), PI 32:0 (7.5%), PI 34:0 (7.3%), and PI 34:2 (7.0%). PI thus contained a significantly higher proportion of saturated acyl chains (53.1%) than PS (29.4%), PE (16.2%), and PC (15.4%). The unusual short-chain substituted PI 28:0 was enriched at the plasma membrane (8.1%, compared with an average of 3.4%). The mitochondrial contact sites, on the other hand, were enriched in a second unusual short-chain containing PI, namely PI 30:0 (9.3%; see ). This enrichment of PI 30:0 in contact sites appeared to be at the expense of PI 34:1, which was reduced to 16.2%, compared with an average of 34.0%. PI 34:1, on the other hand, was enriched in the Golgi membrane (45.1%), but reduced at the plasma membrane (29.4%). PI 34:2 and PI 32:0 were both enriched in the lipid particle membrane.
Overview of Lipid Classes
In general, the four main glycerophospholipid classes, PS, PE, PC, and PI, each were comprised of two main species, which together made up 56–72% of the respective lipid class, and two (or four in the case of PI) minor species that contributed 24–30% to the total. The two main PS species, PS 34:2 and PS 34:1, represented 71.5%, and the two minor species, PS 32:1 and PS 32:2, represented 25.9% of all PS. Similarly, the two major PE species, PE 34:2 and PE 32:2, comprised 61.7%, whereas the two minor species, PE 34:1 and PE 32:1, comprised 30.3% of PE. The PC species were dominated by PC 32:2 and PC 34:2, which together comprised 65.4% and were complemented by the two minor species, PC 32:1 and PC 34:1, which contributed 24.2%. PI contained two major species, PI 34:1 and PI 32:1 (55.8%), and four minor species, PI 32:0, PI 34:0, PI 34:2, and PI 36:1, which together made up 28.7%. Remarkably, not all possible combinations of the four major fatty acids were found esterified to the different glycerophospholipid classes. Disaturated fatty acid-containing species (32:0 and 34:0) were only observed for PI (see ).
Figure 6 Summary showing the average molecular species profile with the corresponding fatty acid composition of each lipid class and their possible interconversion by the de novo pathway from CDP-DAG in the ER, or by contribution from the Kennedy pathway from (more ...)