Abstract
Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph, contains a large number of membrane proteins covalently modified by both saturated and unsaturated fatty acids (Dahl, C.E., Dahl, J.S., and Bloch, K. (1983) J. Biol. Chem. 258, 11814-11818). Pulse-chase experiments show that the radioactivity in the fatty acid moieties of the acyl proteins increases rather than decreases during a 4.5-h incubation period suggesting that a large intracellular pool of metabolites such as phospholipid serves as the donor for protein acylation. We find that cells incubated for 4 h in a growth medium containing [3H]palmitate-labeled phosphatidylglycerol or 2-[3H]palmitoyl dipalmitoylphosphatidylcholine show the same labeling pattern as cells incubated for 4 h in a complete growth medium with [3H]palmitate. Exogenously added phospholipids are not hydrolyzed to free fatty acid during the labeling period. Acylation of proteins is inhibited in cells treated with chloramphenicol showing that there is no pool of proacyl protein in the cell. Labeling of membrane proteins also occurs with [3H]glycerol. Glycerol is incorporated primarily into the same proteins as oleate suggesting that acylation by unsaturated fatty acid may involve a protein bound diglyceride moiety. Palmitate, on the other hand, appears to bind to other sites along the polypeptide chain in addition to the diglyceride moiety.
Highlights
Glycerol is incorporated primarilyinto the same pro- state of the membrane specificallystimulates the synthesis of teins as oleate suggesting that acylationby unsaturated unsaturated phospholipid and in turn, probably indirectly, fatty acidmay involve aproteinbound diglyceride moiety
One of the best studied of this class of modified proteins is the major outer membrane protein of Escherichia coli discovered by BraunandRehn (1) which containsfatty acids ing observations: (1) phospholipid appears to be the direct acyl donor to themembrane proteins(2) acylation of proteins is inhibited in cells treated with chloramphenicol, and (3) [3H]glycerolis incorporated primarily into selected membrane proteins that contain oleic acid
Bovine serumalbumin (Fraction V) andchloramphenicol pears to give rise to the &glyceride-linked fatty acid in the protein (4).In addition, the membrane penicillinase of Bacillus licheniformis has been shown to contain fatty acids and glycerol in linkages analogous to those found in the E. coli protein (5)
Summary
From the James Bryant Conant Laboratories, Departmentof Chemistry, Harvard University, Cambridge, Massachusetts 02138. Pulse-chase experiments show that the radio- Mycoplasma capricolum are covalently modified by saturated activity in the fatty acid moieties of the acyl proteins and unsaturated fattyacids (8,9). A small (synergistic) amount of cholesterol in the membranes stimulates unsaturated phospholipid synthesis and correlates with an increased amount of unsaturated fatty acid bound to certain membrane proteins. Fatty acids covalently bound to proteins have been shown to occur in a wide variety of procaryotic and eucaryotic cells. One of the best studied of this class of modified proteins is the major outer membrane protein of Escherichia coli discovered by BraunandRehn (1) which containsfatty acids ing observations: (1) phospholipid appears to be the direct acyl donor to themembrane proteins(2) acylation of proteins is inhibited in cells treated with chloramphenicol, and (3) [3H]glycerolis incorporated primarily into selected membrane proteins that contain oleic acid. The NH2 terminusof the protein is an N-acylated cysteine
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