Kenneth R. Miller

Oligomeric Rings of the Sec61p Complex Induced by Ligands Required for Protein Translocation

The heterotrimeric Sec61p complex is a major component of the protein-conducting channel of the endoplasmic reticulum (ER) membrane, associating with either ribosomes or the Sec62/63 complex to perform co- and posttranslational transport, respectively. We show by electron microscopy that purified mammalian and yeast Sec61p complexes in detergent form cylindrical oligomers with a diameter of approximately 85 A and a central pore of approximately 20 A. Each oligomer contains 3-4 heterotrimers. Similar ring structures are seen in reconstituted proteoliposomes and native membranes. Oligomer formation by the reconstituted Sec61p complex is stimulated by its association with ribosomes or the Sec62/63p complex. We propose that these cylindrical oligomers represent protein-conducting channels of the ER, formed by ligands specific for co- and posttranslational transport.

Artifacts associated with quick-freezing and freeze-drying

We have studied the structures produced when nonbiological samples were subjected to quick-freezing and freeze-drying with a liquid helium cooled freeze-slamming device. Samples examined in this way included sodium chloride, sucrose, and Tris buffer. A variety of filamentlike and trabeculumlike structures were formed in these preparations. These structures may represent eutectic mixtures formed during the growth of small ice crystals during the freezing process, and exposed during the rapid sublimation of pure ice during the etching process. Samples of biological membranes (isolated chloroplast membranes) were prepared in various buffers by means of this technique. In distilled water, excellent replicas of membrane surfaces were obtained. In salt solutions, however, the membranes appeared to be embedded in a network of thin filaments appearing very much like a cytoskeletal lattice. It is concluded that extreme caution must be used when employing this preparation technique for studies of cell architecture, and that extensive washing of cell components in distilled water may be necessary to obtain faithful representations of cell structure.

Fine structure of the chloroplast membranes ofEuglena gracilis as revealed by freeze-cleaving and deep-etching techniques

SummaryThe chloroplasts ofEuglena gracilis have been examined by freeze-cleaving and deep-etching techniques.The two chloroplast envelope membranes exhibit distinct fracture faces which do not resemble any of the thylakoid fracture faces.Freeze-cleaved thylakoid membranes reveal four split inner faces. Two of these faces correspond to stacked membrane regions, and two to unstacked regions. Analysis of particle sizes on the exposed faces has revealed certain differences from other chloroplast systems, which are discussed. Thylakoid membranes inEuglena are shown to reveal a constant number of particles per unit area (based on the total particle number for both complementary faces) whether they are stacked or unstacked.Deep-etchedEuglena thylakoid membranes show two additional faces, which correspond to true inner and outer thylakoid surfaces. Both of these surfaces carry very uniform populations of particles. Those on the external surface (the A surface) are round and possess a diameter of approximately 9.5 nm. Those on the inner surface (the D surface) appear rectangular (as paired subunits) and measure approximately 10 nm in width and 18 nm in length. Distribution counts of particles show that the number of particles per unit area revealed by freeze-cleaving within the thylakoid membrane approximates closely the number of particles exposed on the external thylakoid surface (the A surface) by deep-etching. The possible significance of this correlation is discussed. The distribution of rectangular particles on the inner surface of the thylakoid sac (D surface) seems to be the same in both stacked and unstacked membrane regions. We have found no correlation between the D surface particles and any clearly defined population of particles on internal, freeze-cleaved membrane faces. These and other observations suggest that stacked and unstacked membranes are similar, if not identical in internal structure.

Unique location of the phycobiliprotein light-harvesting pigment in the Cryptophyceae

The cryptophyte algae, or cryptomonads, comprise a small algal group with a unique photosynthetic apparatus. Both a chlorophyll a/c2 light‐harvesting complex and a phycobiliprotein antenna (which can be either phycoerythrin or phycocyanin) are present, with the phycobiliprotein playing the major role in harvesting light for photosynthesis. Longstanding circumstantial evidence suggested that, in cryptophytes, the phycobiliprotein is located in the intrathylakoid space (thylakoid lumen) rather than on the outer surface of the thylakoid as part of a phycobilisome as in other algae. We used immunogold labeling to show conclusively that 1) the phycoerythrin (PE) of the cryptophyte Rhodomonas lens Pascher and Ruttner is located within the intrathylakoid space, 2) the PE is not exclusively bound to the thylakoid membrane but instead is distributed across the thylakoid lumen and 3) a fraction of this PE is tightly associated with the thylakoid membrane. The thylakoids are not everted to compensate for this unusual arrangement. The location of the major light‐harvesting pigment on the “wrong” side of the otherwise very normal photo‐synthetic membrane is unexpected, unique to the cryptophytes, and, remarkably, does not impair the photosynthetic abilities of this organism. A model is presented which incorporates these results ‐with previous information to give a complete structural picture of the cryptophyte light‐harvesting apparatus.

The ultrastructure of Pyrsonympha and its associated microorganisms

The termite gut flagellates are of interest because of their unusual motile organelles, their ability to digest cellulose, and their symbiotic relationship with prokaryotes inhabiting the insect gut. This report provides a detailed ultrastructural description of Pyrsonympha from the hind‐gut of Reticulitermes flavipes.

Do we really know why chloroplast membranes stack

Abstract Although recent discoveries regarding the phosphorylation of a major light-harvesting protein in the chloroplast membrane have helped to explain the mechanism of membrane stacking, they have failed to explain the underlying reason for the phenomenon. It is suggested that the regulation of excitation energy distribution may not be the reason for membrane adhesion, but rather an adaptive response to stacked membrane organization.

A particle spanning the photosynthetic membrane

The structure of the thylakoid membrane of spinach has been examined by the techniques of freeze-fracturing and deep-etching. Distinct populations of particles exist both within the membrane and on each of its surfaces, as described by other workers. The fact that certain types of particles occasionally form regular lattices in the membrane has been exploited to show that particles visible on both surfaces of the membrane and one fracture face may in fact be merely different views of a single particle that spans the thylakoid membrane. This particle possesses a tetrameric substructure visible on both surfaces of the membrane, but not on the fracture face, indicating that plastic deformation accompanying the fracture process may tend to obscure substructure in intramembrane particles. The existence of a membrane-spanning structure in the photosynthetic membrane raises interesting possibilities concerning the integration of the light reaction within the thylakoid.

Are the photosynthetic membranes of cryptophyte algae inside out

SummaryCryptomonads are unicellular algae with a unique photosynthetic apparatus, both in structure and pigment composition. A cryptomonad,Rhodomonas lens (R. lens), was studied by conventional electron microscopy, freeze-fracture, and freeze-etch in order to determine whether the thylakoids of this alga are everted with respect to those of other plants, as has been postulated (Ganttet al. 1971,Gantt 1979, 1980) as a means to compensate for the location of the cryptomonad light-harvesting apparatus on the opposite side of the thylakoid membrane from that of related algae. We have characterized the thylakoids of this alga and conclude that they are not everted, but are oriented in the same manner as those of other algae and green plants. Implications for energy transfer are discussed.

EFFECTS OF LIGHT AND GLYCEROL ON THE ORGANIZATION OF THE PHOTOSYNTHETIC APPARATUS IN THE FACULTATIVE HETEROTROPH PYRENOMONAS SALINA (CRYPTOPHYCEAE)1

The marine cryptophyte Pyrenomonas salina Santore is capable of autotrophic and heterotrophic nutrition. We studied the physiological and ultrastructural changes that accompany the shift between these nutritional modes. The addition of glycerol to batch cultures of P. salina, grown at an irradiance limiting for photoautotrophic growth, increased its growth rate and induced specific biochemical and structural changes in its photosynthetic system. Results from extracted pigment analyses, thin‐section electron microscopy, and freeze‐fracture electron microscopy indicated that glycerol addition reduced the cell phycoerythrin content, phycoerythrin to chlorophyll a ratio, degree of thylakoid packing, number of thylakoids · cell−1, and PSII particle size. These properties were reduced to a similar extent in cells grown photoautotrophically under an irradiance saturating for growth. These results are consistent with the hypothesis that enhancement of heterotrophic potential occurs at the expense of light‐harvesting ability in glycerol‐grown P. salina.

Heliobacterium chlorum: cell organization and structure.

The basic cellular organization of Heliobacterium chlorum is described using the freeze-etching technique. Internal cell membranes have not been observed in most cells, leading to the conclusion that the photosynthetic apparatus of these organisms must be localized in the cell membrane of the bacterium. The two fracture faces of the cell membrane are markedly different. The cytoplasmic (PF) face is covered with densely packed particles averaging 8 nm in diameter, while the exoplasmic (EF) face contains far fewer particles, averaging approximately 10 nm in diameter. Although a few differentiated regions were noted within these fracture faces, the overall appearance of the cell membrane was remarkably uniform. The Heliobacterium chlorum cell wall is a strikingly regular structure, composed of repeating subunits arranged in a rectangular pattern at a spacing of 11 nm in either direction. We have isolated cell wall fragments by brief sonication in distilled water, and visualized the cell wall structure by negative staining as well as deep-etching.