Freeze-Fracture Electron Microscopy of Thylakoid Membranes

Freeze-Fracture Electron Microscopy of Thylakoid Membranes 

When a replica of the fracture faces is viewed in the transmission electron microscope, such as the electron micrograph made for the barley thylakoid membrane at 100,000X magnification and shown in Eig. 18 (C), the four faces designated as EFu, EFg, PFg and PFu may appear to be side by side on the same plane in the figure, but actually the fracture path jumps from the middle of one membrane to the middle of an adjacent stacked membrane, as seen in Eigs. 18 (B). In reality, the E- and P-faces are separated by a step equal to the thickness ofthe two leaflets, as indicated in the schematic drawing in Pig. 18 (B). 

The four fracture faces in Pig. 18 (C) show imbedded particles of various sizes. In particular, the EFg face shows a profusion of 16 nm-diameter particles. The complementary fracture face PFs contains relatively tightly-packed, but rather indistinct and smaller particles. It is known that fractionation of a thylakoid membrane disrupted by detergent or by mechanical means yields separate granal and stromal fractions. The granal fraction has been found to be enriched in photosystem-II components and activity. 

We now examine some additional results also obtained by freeze-fracture electron microscopy using thylakoid membrane fragments. The membrane fragments were obtained either by detergent fragmentation and subsequent separation by differential centrifugation or by mechanical fragmentation followed by separation by aqueous-polymer two-phase partition. These studies yielded useful and complementary information on the thylakoid-membrane structure itself as well as the functional properties associated with the particular structure. 

A more direct confirmation of the inside-out character of the B3 vesicles was provided by freeze-fracture electron microscopy. Representative replicas ofthe T2 and B3 vesicles are shown in Fig. 19 (D). As expected for the B3 inside-out vesicles, large distinct particles appear in the EF faces next to the intra-thylakoid space [Fig. 19 (D), left], while closely packed small particles appear in the PF faces next to the stroma [Fig. 19 (D), right]. A large number of vesicular faces were examined and the results revealed that on average the B3 fraction contained 74% inside-out and 26% rightside-out vesicles while the T2 fraction contained 89% rightside-out and only 11% inside-out vesicles. 

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VII. Freeze-Fracture Electron Microscopy of Thylakoid Membranes... 

Freeze-fracture electron microscopy of thylakoid membranes has clearly revealed an asymmetric lateral distribution of the various photosynthetic complexes in the granal and stromal membranes, i.e., the distribution of the protein complexes in the membrane is nonrandom. This lateral asymmetry was further substantiated by the results of electron microscopy of the inside-out vesicles discussed in Section Vll. These findings by electron microscopy are summarized by the model shown in Fig. 21 (A). It is a transverse cross section of the thylakoids shown earlier in Fig. 13 (D) and (D ), with the various photosynthetic protein complexes appropriately placed in the granal and stromal regions. 

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