Membranes grow by expansion as new protein and phospholipid components are inser
ID: 206220 • Letter: M
Question
Membranes grow by expansion as new protein and phospholipid components are inserted. Most proteins are inserted as they are synthesized. Phospholipids are synthesized within the plasma membrane by integral membrane proteins (enzymes) of prokaryotes. To determine whether new phospholipids are synthesized in one or both monolayers of the membrane, researchers made use of trinitrobenzenesulfonic acid (TNBS) that reacts specifically with the head group of phosphatidyl-ethanolamine (PE). Because this reagent does not penetrate membranes, it only labels external PE. In the bacterium Bacillus megaterium, TNBS labeling of intact cells shows that 30% of membrane PE is in the external monolayer and 70% is in the cytoplasmic monolayer (and thus not labeled; see figure). You use this technique to examine the distribution of PE synthesized during a 1-minute pulse of 32P-labeled inorganic phosphate which is incorporated into the phospholipid heads. TNBS was added either immediately after the radioactivity or 30 minutes later. The results are shown in the right portion of the figure.
a) Is new PE synthesized in one or both monolayers of the membrane? If only one monolayer, which one? Explain, using specific terms correctly.
b) Given the negligible rate of spontaneous phospholipid flipping on this time scale, explain the different distribution of newly-synthesized PE immediately after the pulse and 30 minutes later.
c) Phosphatidylserine in red blood cells differs from these results in that it appears to reside exclusively in the cytoplasmic monolayer. How would you explain this observation?
Total PE Pulse-labeled PE Chromatographic separation of PE and TNBS-tagged PE 0 min 30 min Tagged with Tagged with TNBS (a) Total PE isolated from intact cells after reaction with excess TNBS. (b) Pulse- labeled PE reacted immediately with TNBS or after a delay of 30 min. Not tagged Not taggedExplanation / Answer
Ans.
a) New PE are synthesized in only one monolayer of the membrane i..e.,cytoplasmic monolayer.
it is evident by the fact that, when TNBS was added immediately after the pulse of 32P-labeled inorganic phosphate, no chromatographic band of PE tagged with TNBS was seen. but after 30 minutes a band is seen which is tagged with TNBS, suggesting that PE is transported to the outer layer after being synthesized in the cytoplasmic layer.
b) as spontaneous phospholipid flipping rate on this time scale is negligible, the different distribution of newly-synthesized PE immediately after the pulse and 30 minutes later can be explained by the activity of the Phospholipid flippases. these proteins are able to translocate phospholipids from one side of a membrane to the other even against a gradient of concentration and thereby able to establish, or annihilate, a transmembrane asymmetrical lipid distribution.
c) PS resides exclusively in the cytoplasmic monolayer of RBCs as it is translocated from the plasma membrane outer monolayer to the inner monolayer by flippase. In erythrocytes, very little lipid metabolism takes place, and no intracellular membranes are present so, new lipids are provided essentially by serum lipoproteins of the blood.