Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane

Abstract

Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells. Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed. Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%). The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of approximately +/- 20%. Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane. Most notably, the plasma membrane is enriched in saturated species of PS and PE. The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further. The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Delta mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species. This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane.

Figure 1

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.

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 against the marker proteins indicated. Hom, cell homogenate; PM, plasma membrane; Nuc, nuclei; LP, lipid particles; Vac, vacuoles; Mit, mitochondria; IM, inner mitochondrial membrane; OM, outer mitochondrial membrane; CS, contact sites; Perox, peroxisomes; 40k, 40,000 g microsomes; 100k, 100,000 g microsomes; Cyt, cytosol.

Figure 3

Morphological analysis of subcellular membrane fractions. Subcellular fractions were isolated as outlined in Fig. 1, fixed, and processed for EM as described in Materials and Methods. PM, plasma membrane; LP, lipid particles; Vac, vacuoles; Mit, mitochondria; IM, inner mitochondrial membrane; OM, outer mitochondrial membrane; Perox, peroxisomes; 40k, 40,000 g microsomes. Bars, 500 nm.

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 are color coded: PA, magenta; PE, blue; PS, cyan; PI, red; MMPE, lilac; sphingolipids, black. Positions of major molecular species are indicated. PA, phosphatidic acid; PS, phosphatidylserine; PE, phosphatidylethanolamine; MMPE, monomethylphosphatidylethanolamine; PI, phosphatidylinositol; Cer, ceramide C; IPC, inositolphosphorylceramide; M(IP)₂C, mannosyldiinositolphosphorylceramide. The total carbon chain length (x) and number of carbon–carbon double bonds (y) of individual lipid molecular species is specified (x:y).

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 wild-type (EMY30) and elo3Δ (EMA40) mutant cells. Peak assignment to PS molecular species is indicated.

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 local pools of DAG in the Golgi apparatus. The two major molecular species of each lipid class are boxed in gray. Linear increases and decreases in the relative abundance of 32:1 and 34:1 containing species along the biosynthetic route from PS via PE to PC are indicated by the triangles.