Cellulose-synthesizing terminal complexes and microfibril structure in the brown alga Sphacelaria rigidula (Sphacelariales,Phaeophyceae)*

The brown alga Sphacelaria rigidula Kützing synthesizes cellulose microfibrils as determined by CBH I-gold labeling. The cellulose microfibrils are thin, ribbon-like structures with a uniform thickness of about 2.6 nm and a variable width in the range of 2.6-30 nm. Some striations appear along the longitudinal axis of the microfibrils. The developed cell wall in Sphacelaria is composed of three to four layers, and cellulose micro-fibrils are deposited in the third layer from the outside of the wall. A freeze fracture investigation of this alga revealed cellulose-synthesizing terminal complexes (TCs), which are associated with the tip of microfibril impressions in the plasmatic fracture face of the plasma membrane. The TCs consist of subunits arranged in a single linear row. The average diameter of the sub-units is about 6 nm, and the intervals between the neighboring subunits, about 9 nm, are relatively constant. The number of subunits constituting the TC varies between 10 and 100, so that the length of the whole TC varies widely. A model that has been proposed for the assembly of thin, ribbon-like microfibrils was applied to microfibril assembly in Sphacelaria.     

Immunogold labeling of rosette terminal cellulose-synthesizing complexes in the vascular plant vigna angularis

The catalytic subunit of cellulose synthase is shown to be associated with the putative cellulose-synthesizing complex (rosette terminal complex [TC]) in vascular plants. The catalytic subunit domain of cotton cellulose synthase was cloned using a primer based on a rice expressed sequence tag (D41261) from which a specific primer was constructed to run a polymerase chain reaction that used a cDNA library from 24 days postanthesis cotton fibers as a template. The catalytic region of cotton cellulose synthase was expressed in Escherichia coli, and polyclonal antisera were produced. Colloidal gold coupled to goat anti-rabbit secondary antibodies provided a tag for visualization of the catalytic region of cellulose synthase during transmission electron microscopy. With a freeze-fracture replica labeling technique, the antibodies specifically localized to rosette TCs in the plasma membrane on the P-fracture face. Antibodies did not specifically label any structures on the E-fracture face. Significantly, a greater number of immune probes labeled the rosette TCs (i.e., gold particles were 20 nm or closer to the edge of the rosette TC) than did preimmune probes. These experiments confirm the long-held hypothesis that cellulose synthase is a component of the rosette TC in vascular plants, proving that the enzyme complex resides within the structure first described by freeze fracture in 1980. In addition, this study provides independent proof that the CelA gene is in fact one of the genes for cellulose synthase in vascular plants.     

The assembly of cellulose microfibrils in Valonia macrophysa Kütz

The assembly of cellulose microfibrils was investigated in artificially induced protoplasts of the alga, Valonia macrophysa (Siphonocladales). Primary-wall microfibrills, formed within 72 h of protoplast induction, are randomly oriented. Secondary-wall lamellae, which are produced within 96 h after protoplast induction, have more than three orientations of highly ordered microfibrils. The innermost, recently deposited micofibrils are not parallel with the cortical microtubules, thus indicating a more indirect role of microtubules in the orientation of microfibrils. Fine filamentous structures with a periodicity of 5.0–5.5 nm and the dimensions of actin were observed adjacent to the plasma membrane. Linear cellulose-terminal synthesizing complexes (TCs) consisting of three rows, each with 30–40 particles, were observed not only on the E fracture (EF) but also on P fracture (PF) faces of the plasma membrane. The TC appears to span both faces of the bimolecular leaflet. The average length of the TC is 350 nm, and the number of TCs per unit area during primary-wall synthesis is 1 per m². Neither paired TCs nor granule bands characteristic of Oocystis were observed. Changes in TC structure and distribution during the conversion from primary- to secondary-wall formation have been described. Cellulose microfibril assembly in Valonia is discussed in relation to the process among other eukaryotic systems.Abbreviations TC terminal complex - EF E (outer leaflet) fracture face of the plasma membrane - PF P (inner leaflet) fracture face of the plasma membrane - MT microtubule - PS protoplasmic surface of the membrane     

Occurrence of the putative microfibril-synthesizing complexes (linear terminal complexes) in the plasma membrane of the epiphytic marine red algaErythrocladia subintegra Rosenv.

Summary Cells of thalli at different developmental stages of the epiphytic marine red algaErythrocladia subintegra have been studied by freeze-etching. It was found that the plasma membrane exhibits linear microfibril-termnal synthesizing complexes (TCs), randomly distributed consisting of four rows of linearly-arranged particles (average diameter of particles 8.6 nm); each row of TCs consists of 5–33 particles (average 15). The TCs were observed on both fracture faces (PF and EF) but more clearly on the PF face. These structures appear to span both the outer and inner leaflets of the plasma membrane (transmembrane complexes)-The TCs have stable width (35 nm) and vary in length (41–311 nm, average 181 nm). The TCs subunits are highly ordered arrays forming a semicylinder. The average density of TCs on the PF face is 5.5TC/m². The microfibrils are randomly distributed and have a mean width of 39.4 nm (ranging from 16 to 70 nm). Many TCs are associated with the ends of microfibrils and microfibril imprints. The structural characteristics of linear TCs in the red algaErythrocladia are compared with those of the so far investigated Chlorophyta spp. All results favour the suggestion that TCs in the plasma membrane ofErythrocladia cells are involved in the biosynthesis, assembly and orientation of microfibrils.     

High resolution analysis of the formation of cellulose synthesizing complexes inVaucheria hamata

Summary Ultrastructure and assembly of cellulose terminal synthesizing complexes (terminal complexes, TCs) in the algaVaucheria hamata (Waltz) were investigated by high resolution analytical techniques for freeze-fracture replication.Vaucheria TCs consist of many diagonal rows of subunits located on the inner leaflet of the plasma membrane. Each row contains about 10–18 subunits. The subunits themselves are rectangular, approx. 7×3.5 nm, and each has a single elliptical hole which may be the site of a single glucan chain polymerization. The subunits are connected with extremely small filaments (0.3–0.5 nm). Connections are more extensive in a direction parallel to the subunit rows and less extensive perpendicular to them. Nascent TC subunits are found to be packed within globules (15–20 nm in diameter) which are larger than typical intramembranous particles (IMPS are 10–11 nm in diameter) distributed in the plasma membrane. The subunits in the globule, which may be a zymogenic precursor of the TC, are generally exhibited in the form of doublets. Approximately 6 doublets are connected to a center core with small filaments. The globules are inserted into the plasma membrane together with IMPS by the fusion of cytoplasmic (Golgi derived) vesicles. Two or three globules attach to each other, unfold, and expand to form the first subunit rows of the TC on the inner leaflet of the plasma membrane. More globules attach to the structure and unfold until the nascent TC consists of a few rows of subunits. These rows are arranged almost parallel to each other. Two formation centers of subunits appear at both ends of an elongating TC. New subunits carried by the globules are added at each of these centers to create new rows until the elongating TC structure is completed. On the basis of this study, a model of TC assembly and early initiation of microfibril formation inVaucheria is proposed.Abbreviations IMPS intramembranous particles - MF microfibril - TC terminal complex     

TIP CELL GROWTH AND THE FREQUENCY AND DISTRIBUTION OF CELLULOSE MICROFIBRIL-SYNTHESIZING COMPLEXES IN THE PLASMA MEMBRANE OF APICAL SHOOT CELLS OF THE RED ALGA PORPHYRA YEZOENSIS1

The supramolecular organization of the plasma membrane of apical cells in shoot filaments of the marine red alga Porphyra yezoensis Ueda (conchocelis stage) was studied in replicas of rapidly frozen and fractured cells. The protoplasmic fracture (PF) face of the plasma membrane exhibited both randomly distributed single particles (with a mean diameter of 9.2 ± 0.2 nm) and distinct linear cellulose microfibril-synthesizing terminal complexes (TCs) consisting of two or three rows of linearly arranged particles (average diameter of TC particles 9.4 plusmn; 0.3 nm). The density of the single particles of the PF face of the plasma membrane was 3000 μm^?2, whereas that of the exoplasmic fracture face was 325 μm^?2. TCs were observed only on the PF face. The highest density of TCs was at the apex of the cell (mean density 23.0 plusmn; 7.4 TCs μm^?2 within 5 μm from the tip) and decreased rapidly from the apex to the more basal regions of the cell, dropping to near zero at 20 μm. The number of particle subunits of TCs per μm² of the plasma membrane also decreased from the tip to the basal regions following the same gradient as that of the TC density. The length of TCs increased gradually from the tip (mean length 46.0 plusmn; 1.4 nm in the area at 0–5 μm from the tip) to the cell base (mean length 60.0 plusmn; 7.0 μm in the area at 15–20 μm). In the very tip region (0–4 μm from the apex), randomly distributed TCs but no microfibril imprints were observed, while in the region 4–9 μm from the tip microfibril imprints and TCs, both randomly distributed, occurred. Many TCs involved in the synthesis of cellulose microfibrils were associated with the ends of microfibril imprints. Our results indicate that TCs are involved in the biosynthesis, assembly, and orientation of cellulose microfibrils and that the frequency and distribution of TCs reflect tip growth (polar growth) in the apical shoot cell of Porphyra yezoensis. Polar distribution of linear TCs as “cellulose synthase” complexes within the plasma membrane of a tip cell was recorded for the first time in plants.     

Cellulose microfibril assembly inErythrocladia subintegra Rosenv.: An ideal system for understanding the relationship between synthesizing complexes (TCs) and microfibril crystallization

Summary The marine red algaErythrocladia subintegra synthesizes cellulose microfibrils as determined by CBH I-gold labelling, X-ray and electron diffraction analyses. The cellulose microfibrils are quite thin, ribbon-like structures, 1–1.5 nm in thickness (constant), and 10–33 nm in width (variable). Several laterally associated minicrystal components contribute to the variation in microfibrillar width. Electron diffraction analysis suggested a uniplanar orientation of the microfibrils with their (101) lattice planes parallel to the plasma membrane surface of the cell. The linear particle arrays bound in the plasma membrane and associated with microfibril impressions recently demonstrated inErythrocladia have been shown in this study to be the cellulose-synthesizing terminal complexes (TCs). The TCs appear to be organized by a repetition of transverse rows consisting of four TC subunits, rather than by four rows of longitudinallyarranged TC subunits. The number of transverse rows varied between 8–26, corresponding with variation in the length of the TCs and the width of the microfibrils. The spacings between the neighboring transverse rows are almost constant being 10.5–11.5 nm. Based on the knowledge thatAcetobacter, Vaucheria, andErythrocladia synthesize similar thin, ribbon-like cellulose microfibrils, the structural characteristics common to the organization of distinctive TCs occurring in these three organisms has been discussed, so that the mode of cellulose microfibril assembly patterns may be deciphered.     

Cellulose synthesizing terminal complexes and morphogenesis in tip-growing cells of Syringoderma phinneyi (Phaeophyceae)

The intramembrane particles and cellulose synthesis of the brown alga Syringoderma phinneyi Henry et Müller were examined using replicas of freeze‐fractured apical cells. Like in other brown algae, linear terminal complexes (TCs) were found in the plasmatic fracture face (PF) of the plasmalemma, which are the putative cellulose synthases. Terminal complexes consist of a single row of particles, each particle composed of two sub‐units, and are found in close relationship with cellulose microfibril imprints. Examination of the distribution of TCs revealed a clear apico‐basal gradient, with a higher density of TCs in the apical part. This seems to reflect the tip growth of the apical cells. The rate of cellulose synthesis per TC subunit was calculated based on the dimensions of the TCs and cellulose microfibrils.     

Freeze-fracture studies in the brown algaAsteronema rhodochortonoides

Summary The gross structure of the cell wall and the organization of the plasmalemma of the filamentous brown algaAsteronema rhodochortonoides were examined in replicas of freeze-fractured cells. The protoplasmic fracture face (PF) of the plasmalemma, apart from the single particles, exhibits two particular particle complexes, i.e., single linear arrays of closely packed particles, and well defined particle pentads. The former display a consistent relationship with the ends of microfibril imprints and therefore are considered as terminal complexes (TCs). They seem to be composed of subunits, each one consisting of two particles. The average diameter of the particles is 7 nm. The number of the subunits forming the TCs varies between 2 and 40. Short TCs, consisting of 3–5 subunits were also found on the PF of dictyosome vesicles, a fact suggesting the involvement of the Golgi apparatus in exocytosis of preformed TC portions. The occurrence, distribution and size of the TCs appear to be related to the developmental stage of the cell. A large number of TCs occur in actively growing cells, while a few or no TCs are found in differentiated cells. The pentads are rectangular structures consisting of five particles, four in the corners and one in the centre. Their dimensions are very constant, but their occurrence and distribution varies. They occur in young developing cells where TCs are few or absent, but were also observed in areas showing many TCs. In differentiated cells no pentads were found. Pentad-like structures were rarely observed on the PF of dictyosome vesicles or cisternae. The observations support the hypothesis that pentads are involved in the synthesis of matrix polysaccharides, which are the major components of brown algal cell wall and their synthesis begins before that of cellulose.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Endocytosis by human platelets: metabolic and freeze-fracture studies

The mechanism by which platelets endocytose or release particulate or soluble substances is poorly understood. Engulfed materials enter the open canalicular system (OCS) by a process akin to phagocytosis, but fusion of platelet granules with the OCS is rarely observed. Secretion of granule contents, a concomitant of the "release reaction" which occurs during platelet aggregation, does not take place by extrusion at the surface membrane as is true for other secretory cells. Some substances may be secreted without obvious granule loss. To examine whether structural properties of the platelet membrane could account for this unusual behavior, thin section and freeze-fracture analyses were performed on platelets which had undergone endocytosis under a variety of experimental conditions. After freeze-cleavage, most of the intramembranous particles (IMP) remain associated with the outer leaflet of the platelet plasma membrane. The sites where the OCS reaches the surface membrane are marked by pits on the cytoplasmic leaflet (P face) and by complementary protrusions on the outer leaflet (E face) of the membrane. Endocytosis of small particles and solutes takes place via these structures. This process is not energy dependent but arrested at 4 degrees C. Distension of the OCS does not appear to affect the size or number of the pits. On the other hand, large particles are taken up by membrane invagination without redistribution of IMP's and independent of the pits. This process is sensitive to metabolic inhibition. Thus, the studies have demonstrated the existence of two different pathways for platelet endocytosis which are postulated to be also involved in secretion. The selective release of substances contained in different granules may be related to the "inside-out" structure of the plasma and OCS membranes.     

Assembly of complement components C5b-8 and C5b-9 on lipid bilayer membranes: visualization by freeze-etch electron microscopy

We have visualized by freeze-etch electron microscopy the macromolecular complexes of complement, C5b-8 and C5b-9, respectively, assembled on synthetic phospholipid bilayers. These complexes were formed sequentially by using purified human complement components C5b-6 followed by C7, C8, and C9. Complexes of C5b-8 were observed on the external surface (ES) of vesicles as 12-nm particles that tended to form polydisperse aggregates. The aggregates were sometimes of a regular chainlike structure containing varying numbers of paired subunits. Etching of vesicles containing C5b-9 complexes revealed on the ES large rings of approximately 27-nm outer diameter. One or two knobs usually were attached to the perimeter of the rings. Splitting of the membrane resulted in partitioning of the C5b-9 with the outer leaflet. Thus, round holes of approximately 17-nm diameter were present in the protoplasmic face (PF), and raised circular stumps of a matching size were present on the exoplasmic face (EF) of C5b-9 vesicles. C5b-9 complexes were frequently localized in regions of the lowest lipid order. That is, in micrographs of the EF and ES, single C5b-9 complexes were located where the ripples of the P beta' phase bend or reach a dead end, and linear arrays of C5b-9 complexes outlined disclination-like structures in the lattice; the holes in the PF mirrored this distribution. The membrane immediately surrounding C5b-9 rings was often sunk inwardly over an area much larger than that of the ring itself.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure and associations of the double S layer on the cell wall of Aquaspirillum sinuosum

Aquaspirillum sinuosum cell walls bear two paracrystalline, proteinaceous surface layers (S layers). Each shows a different symmetry: the inner layer is closely apposed to the outer membrane and is a tetragonal array (90 degrees axes; 5-nm units; repeat frequency 8 nm); the outer layer is a hexagonal array on the external surface (14-nm units; repeat frequency 18 nm) and, although the units have a six-pointed stellate form, the linkage between units is not resolved. The outer layer consists of a major 130-kDa protein and a 180-kDa minor component; these co-extract, co-assemble, and are inseparable by hydroxylapatite chromatography or by recrystallization. The solubilizing effects of reagents suggest stabilization by hydrogen bonding and Ca2+. The two outer layer proteins are serologically related and show partial identity by peptide mapping. Periodic acid--Schiff staining of the 180-kDa band suggests that this may be a glycosylated form of the 130-kDa component. The inner layer components form a doublet of 75- and 80-kDa polypeptides with extreme resistance to extraction. Close apposition to the outer membrane, resistance to chaotropes, aqueous insolubility, and behaviour in charge-shift electrophoresis suggest hydrophobic interaction between subunits and an integral association with the outer membrane.     

Localization of a Porphyromonas gingivalis 26-kilodalton heat-modifiable, hemin-regulated surface protein which translocates across the outer membrane.

We recently identified a 26-kDa hemin-repressible outer membrane protein (Omp26) expressed by the periodontal pathogen Porphyromonas gingivalis. We report the localization of Omp26, which may function as a component of a hemin transport system in P. gingivalis. Under hemin-deprived conditions, P. gingivalis expressed Omp26, which was then lost from the surface after a shift back into hemin-rich conditions. Experiments with 125I labeling of surface proteins to examine the kinetics of mobilization of Omp26 determined that it was rapidly (within less than 1 min) lost from the cell surface after transfer into a hemin-excess environment. When cells grown under conditions of hemin excess were treated with the iron chelator 2,2'-bipyridyl, Omp26 was detected on the cell surface after 60 min. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses using purified anti-Omp26 monospecific polyclonal immunoglobulin G antisera established that Omp26 was heat modifiable (39 kDa unheated) and consisted of a single protein species. Immunogold labeling of negatively stained and chemically fixed thin-section specimens indicated that Omp26 was associated with the cell surface and outer leaflet of the P. gingivalis outer membrane in hemin-deprived conditions but was buried in the deeper recesses of the outer membrane in hemin-excess conditions. Analysis of subcellular fractions of P. gingivalis grown either in hemin-excess or hemin-deprived conditions detected Omp26 only in the cell envelope fraction, not in the cytoplasmic fraction or culture supernatant. Limited proteolytic digestion of hemin-deprived P. gingivalis with trypsin and proteinase K verified the surface location of Omp26 as well as its susceptibility to proteolytic digestion. Heat shock treatment of hemin-excess-grown P. gingivalis also resulted in Omp26 translocation onto the outer membrane surface even in the presence of hemin. Furthermore, hemin repletion of heat-shocked, hemin-deprived P. gingivalis did not result in Omp26 translocation off the outer membrane surface, suggesting that thermal stress inactivates this transmembrane event. This newly described outer membrane protein appears to be associated primarily with the outer membrane, in which it is exported to the outer membrane surface for hemin binding and may be imported across the outer membrane for intracellular hemin transport.     

Aminophospholipid translocation in erythrocytes: evidence for the involvement of a specific transporter and an endofacial protein

The transport of exogenously supplied fluorescent analogues of aminophospholipids from the outer to inner leaflet in red blood cells (RBC) is dependent upon the oxidative status of membrane sulfhydryls. Oxidation of a sulfhydryl on a 32-kDa membrane protein by pyridyldithioethylamine (PDA) has been previously shown [Connor & Schroit (1988) Biochemistry 27, 848-851] to inhibit the transport of NBD-labeled phosphatidylserine (NBD-PS). In the present study, other sulfhydryl oxidants were examined to determine whether additional sites are involved in the transport process. Our results show that diamide inhibits the transport of NBD-PS via a mechanism that is independent of the 32-kDa site. This is shown by the inability of diamide to block labeling of the 32-kDa sulfhydryl with 125I-labeled PDA and to protect against PDA-mediated inhibition of NBD-PS transport. diamide-mediated inhibition, but not PDA-mediated inhibition, could be reversed by reduction with cysteamine or endogenous glutathione. Similarly, treatment of RBC with 5,5'-dithiobis(2-nitrobenzoic acid), which depletes endogenous glutathione and induces oxidation of endofacial proteins [Reglinski et al. (1988) J. Biol. Chem. 263, 12360-12366], inhibited NBD-PS transport in a manner analogous to diamide. Once established, the asymmetric distribution of NBD-PS could not be altered by oxidation of either site. These data indicate that a second site critical to the transport of aminophospholipids resides on the endofacial surface and suggest that the transport of aminophospholipids across the bilayer membrane of RBC depends on a coordinated and complementary process between a cytoskeletal component and the 32-kDa membrane polypeptide; both must be operative for transport to proceed.     

Bidirectional transbilayer movement of phospholipid analogs in human red blood cells. Evidence for an ATP-dependent and protein-mediated process.

The transbilayer movement of fluorescent and isotopically labeled analogs of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) from the outer to the inner leaflet (flip) and from the inner to the outer leaflet (flop) of human red blood cells (RBC) was examined. The inward movement of 1-oleoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole-aminocaproyl)- (C6-NBD-), 1-oleoyl-2-(N-(3-(3-[125I]iodo-4-hydroxyphenyl)propionyl)aminocaproyl)- (C6-125I-), or 1-oleoyl-2-(N-(3-3-[125I]iodo-4-azido-phenyl)propionyl)aminocaproyl- (C6-125I-N3-) analogs of PC and PE were relatively slow. In contrast, all analogs of PS and PE analogs containing aminododecanoic acid (C12 lipids) were rapidly transported to the cell's inner leaflet. Analysis of 125I-N3 lipids cross-linked to membrane proteins revealed labeling of 32-kDa Rh polypeptides that was dependent on the lipid's capacity to be transported to the inner leaflet but was independent of lipid species. To investigate whether lipids could also be transported from the inner to the outer leaflet, lipid probes residing exclusively in the inner leaflet were monitored for their appearance in the outer leaflet. Lipid movement could not be detected at 0 degrees C. At 37 degrees C, however, approximately 70% of the PC, 40% of the PE, and 15% of the PS redistributed to the cells outer leaflet, thereby attaining their normal asymmetric distribution. Continuous incubation in the presence of bovine serum albumin depleted the cells of the analogs (t1/2 approximately 1.5 h) in a manner that was independent of lipid species. Similar to the inward movement of aminophospholipids, the outward movement of PC, PE, and PS was ATP-dependent and could be blocked by oxidation of membrane sulfhydryls and by the histidine reagent bromophenacyl bromide. Evidence is presented which suggests that the outward movement of lipids is an intrinsic property of the cells unrelated to compensatory mechanisms due to an imbalance in lipid distribution.     

Freeze-fracture study of the zymogen granule membrane of pancreas: two novel types of intramembrane particles

The ultrastructure of the zymogen granule (ZG) membrane has been observed in vitro by rapid freezing and freeze-fracture techniques. Unidirectional shadowing of the plasmic fracture (PF) leaflet of the intact granule reveals a relatively smooth surface uniformly studded by intramembrane particles (IMP; 360 microns2) their diameters ranging from 5 to 18 nm (mean = 10.2 nm) but does not allow a clear visualization of the particles on the external fracture (EF) leaflet. Indeed, rotary shadowing reveals that the EF leaflet presents a highly textured subparticle background with a significantly lower frequency of IMP (44 microns2) showing diameters from 9 to 18 nm and a shift to larger IMP (mean = 12.3 nm). Two hitherto undescribed types of IMP are found on both leaflets of the membrane: first a population of 13-nm particles with an electron-lucent center or "pore", the most frequent type on the EF face (26%), is a second population of large IMP (15 nm) characterized by a large "pore" (5.0 nm diameter) subdivided by a delicate cross-shaped structure. In alkaline conditions, pH 8.2, ZG lysis occurs rapidly and membrane ghosts thus obtained were rapidly frozen or suspended in dextran and filtered immediately. Transmission electron microscopy (TEM) shows many opened ghosts with adhering amorphous material and numerous small vesicles near or still attached to openings in the ghosts. Freeze-fracture preparations show that granule lysis is accompanied by major alterations of membrane ultrastructure; the subparticle background on the EF leaflet is now visible only as a cap or linear crest at one pole of the ghosts. These two newly formed zones are demarcated by a row of 13-nm particles, whereas the other IMP are confined to the subparticle background. Some images suggest that the subparticle background and 13-nm IMP necklace give rise to vesicles, some of them occasionally attached to the ghosts. The subparticle background on the EF leaflet shows a complementary imprint on the PF leaflet which is similarly modified. This study shows the presence of hitherto undescribed types of IMP and also demonstrates alterations of certain domains of zymogen granule membranes that occur at the moment of lysis, associated with a redistribution of different particle populations.     

Direct evidence for interaction between COOH-terminal regions of cytochrome b558 subunits and cytosolic 47-kDa protein during activation of an O(2-)-generating system in neutrophils.

Cytochrome b558 in phagocytes is a transmembrane protein composed of large and small subunits and considered to play a key role in O2- generation during the respiratory burst. The COOH-terminal regions of the cytochrome subunits protrude to the cytoplasmic side and are assumed to be the sites for association with cytosolic components to form an active O(2-)-generating complex (Imajoh-Ohmi, S., Tokita, K., Ochiai, H., Nakamura, M., and Kanegasaki, S. (1992) J. Biol. Chem. 267, 180-184). We show here that two synthetic peptides corresponding to the COOH-terminal region of each subunit inhibit NADPH-dependent oxygen uptake induced by sodium dodecyl sulfate (SDS) in a cell-free system consisting of plasma membrane and cytosol. The inhibition was observed when either peptide was added to the system before, but not after, the activation with SDS suggesting that interaction between the COOH-terminal regions of the cytochrome subunits and cytosolic components is important for the assembly and the activity of the O(2-)-generating system. Using the cross-linking reagent dimethyl 3,3'-dithiobis-propionimidate, we found that the cytosolic 47-kDa protein, an essential component of the O(2-)-generating system, interacted with the synthetic peptides in the presence of SDS. In addition to the 47-kDa protein, a 17-kDa protein was found to be associated with the peptide corresponding to the COOH-terminal region of the small subunit. These results indicate that the cytosolic COOH-terminal regions of cytochrome b558 subunits are the binding sites for both the cytosolic 47-kDa protein and the 17-kDa protein and that the binding takes place during activation of the system.     

Identification of the uridine 5'-diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc

Photoaffinity labeling of purified cellulose synthase with [beta-32P]5-azidouridine 5'-diphosphoglucose (UDP-Glc) has been used to identify the UDP-Glc binding subunit of the cellulose synthase from Acetobacter xylinum strain ATCC 53582. The results showed exclusive labeling of an 83-kDa polypeptide. Photoinsertion of [beta-32P]5-azido-UDP-Glc is stimulated by the cellulose synthase activator, bis-(3'----5') cyclic diguanylic acid. Addition of increasing amounts of UDP-Glc prevents photolabeling of the 83-kDa polypeptide. The reversible and photocatalyzed binding of this photoprobe also showed saturation kinetics. These studies demonstrate that the 83-kDa polypeptide is the catalytic subunit of the cellulose synthase in A. xylinum strain ATCC 53582.     

Assembly of Cellulose Synthesizing Complexes on the Plasma Membrane of Boodlea coacta

The assembly of cellulose synthesizing complexes (terminal complexes,TCs) on the plasma membrane of Boodlea coacta was investigatedduring the formation of both the matrix-rich layer (MRL) andfibril-rich layers (FRLs) of cell walls. The TCs appeared tobe located mostly within the outer leaflet of the plasma membrane,and were observed as elliptical protrusions consisting of manyparticles of about 9 nm in diameter. Their length varied from100 to 500 nm (average, 220 nm) during MRL formation and from100 to 860 nm (average, 360 nm) during FRL formation. A correlationwas found between the length of TCs and the microfibril widthin both MRL and FRL. On the E-face of the plasma membrane, numerous round protrusions(30–130 nm in diameter), consisting of many particles,8–10 nm in diameter, were also present. Their densitywas greater during FRL formation than during MRL formation.Some of these structures larger than 100 nm were associatedwith microfibril impressions and some appeared to be bound tothe TCs. These protrusions increased in number with Calcofluortreatment but decreased in number when the dye was removed fromthe culture medium. Thus, the TCs may be assembled from massesof particles aggregated on the outer surface of the plasma membrane,and may grow longer by incorporation of these masses. The appearanceof the longer TCs during FRL formation is probably due to thegreater density of these masses. (Received May 1, 1985; Accepted August 16, 1985)     

SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application

 Recently, we have developed a quick-freezing/freeze-fracture replica labeling technique, sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL), to study the two-dimensional distribution of cytochemical labeling on the membrane surface and the relationship of this distribution to images of freeze-fracture replicas created by platinum shadowing. In SDS-FRL, unfixed, quick-frozen cells, after freeze-fracture and platinum/carbon shadowing, are treated with SDS. The detergent dissolves unfractured areas of the cell membranes, with the release of the cytoplasmic contents. The cytoplasmic and exoplasmic membrane surfaces can be then labeled cytochemically. Integral membrane proteins, revealed as intramembrane particles by freeze-fracture replication, which are indistinguishable on a purely morphological basis, can be selectively labeled by SDS-FRL with specific antibody. In addition, this approach can be applied to examine the transmembrane phospholipid distribution in various cell and intracellular membranes. In this review, we describe the practical procedure for SDS-FRL in detail, present its application to labeling of various membrane components, and briefly discuss the possibility of a combination of SDS-FRL with atomic force microscopy. Accepted: 1 November 1996