Massive ribonucleoprotein bodies in gametophyte vegetative cells of the mossTimmiella barbuloides (Brid.) Moenk

Summary Vegetative cells of the gametophyte phase of the mossTimmiella barbuloides (Pottiales) are characterized by large cytoplasmic bodies of spherical shape (SBs) whose ribonucleoprotein composition is cytochemically demonstrated. SBs seem to be derived from massive aggregation of cytoplasmic ribosomes, with possible participation by rough endoplasmic reticulum elements. SBs have been found in stereids, parenchymatous cells and young hydroids of the gametophyte stem, and in euricysts of the leaf nerve. The SBs develop early in the course of cell differentiation and, once formed, persist until advanced stages of cell senescence.     

Formation of the plasma membrane during regeneration ofParamecium caudatum

Summary Fragments ofParamecium caudatum cells obtained by merotomy were fixed in 1% OsO₄ within 5 seconds after cutting. The ultrastructure of the damaged area of the fragment was studied in oriented ultrathin sections and by scanning electron microscopy. The cytoplasm exposed by merotomy was covered during a few seconds with a new membrane. This was a typical trilaminar membrane continuous with the plasma membrane covering the undamaged surface of the cell. The surface over the wound was wrinkled into irregular grooves and ridges. The cytoplasm, mitochondria and trichocysts in the injured region were electron translucent. The cytoplasm under the new membrane contained an unusually high amount of small membrane vesicles, 20–90 nm in diameter. These were probably the remnants of subpellicular alveoli and the plasma membrane destroyed by microsectioning. The possibility that the exposed cytoplasm would be covered by mere shifting of the existing plasma membrane can be excluded. The complex structure of the cortex with its subpellicular alveoli and regularly distributed cilia provide a strong argument against this notion. It seems probable that the new membrane was built up from the available molecular material,e.g., phospholipids and proteins present in the cytoplasm. Fragments of the membrane and alveolar membranes in the form of small vesicles may have also been included into the new membrane.     

Lateral enzyme topology in the rough endoplasmic reticulum of rat liver

Summary Rough microsomes were subfractionated on the basis of different properties in order to investigate the nature and extent of the enzyme heterogeneity of these vesicles. A discontinuous gradient, containing monovalent cations allowed the separation of a ribosome-poor membrane fraction which was enriched in electron transport enzymes and relatively poor in phosphatases. Zonal centrifugation on a stabilizing gradient separated 3 fractions characterized by enrichment of electron transport enzymes, glucose-6-phosphatase and adenosinetriphosphatase, respectively. An essentially similar pattern was seen when ribosomes were removed with EDTA and the denuded vesicles subfractionated on a sucrose gradient. Rough microsomes from phenobarbitaltreated rats exhibited the same pattern both qualitatively and quantitatively. It appears that electron transport enzymes and two types of phosphatases are heterogeneously distributed among rough microsomal vesicles.This work has been supported by grants from the Swedish Medical Research Council. The authors wish to thank Mrs. Ulla-Britta Torndal for her valuable technical assistance     

Characterization of peroxisomes in glucose-grown Hansenula polymorpha and their development after the transfer of cells into methanol-containing media

Cells of Hansenula polymorpha growing exponentially on glucose generally contained a single peroxisome of small dimension, irregular in shape and located in close proximity to the cell wall. Crystalline inclusions in the peroxisomal matrix were not observed. Associations of the organelles with one or more strands of endoplasmic reticulum were evident. In stationary phase cells the size of the peroxisomes had increased considerably. They were more cubical in form and showed a partly or completely crystalline matrix.After the transfer of cells growing exponentially on glucose into media containing methanol, large peroxisomes with a partly crystalline matrix developed in the cells within 6 h. These organelles originated from the small peroxisomes in the glucose-grown cells. De novo synthesis of peroxisomes was not observed. Prolonged cultivation in the presence of methanol resulted in a gradual increase in the number of peroxisomes by means of separation of small peroxisomes from mature organelles. During growth of peroxisomes associations with the endoplasmic reticulum remained evident.The increase in volume density of peroxisomes in stationary phase cells grown on glucose and in methanol-grown cells was accompanied by the synthesis of the peroxisomal enzymes alcohol oxidase and catalase. Cytochemical staining techniques revealed that alcohol oxidase activity was only detected when the peroxisomes contained a crystalloid inclusion. Since in peroxisomes of an alcohol oxidase-negative mutant of Hansenula polymorpha crystalline inclusions were never detected, it is concluded that the development of crystalloids inside peroxisomes is due to the accumulation of alcohol oxidase in these organelles.     

The isolation of two new lanostane triterpenoid derivatives from the edible mushroom Astraeus asiaticus

Two lanostane triterpenoid derivatives, astrasiaone (2), (22S, 25R, 26R)-26-methoxy-22-26-epoxylanost-8-en-3-one, and astrasiate (3), (3α, 22S, 25R)-3, 22-dihydroxylanost-8-en-26 -oate, together with six known compounds, astraodorol (1), artabotryol B (4), artabotryol C1 (5), 6-dehydrocerevisterol (6), ergosterol (7) and hypaphorine (8) were isolated from the edible mushroom Astraeus asiaticus. 3 and 4 exhibited weak cytotoxicity against KB and NCI-H187 cancer cell lines. A comparison of the structures of 2 and 3 to that of 1, 4 and 5 suggest that these two new compounds could be the intermediate form that occurs during biogenesis.     

Crystal Structure of BamB Bound to a Periplasmic Domain Fragment of BamA,the Central Component of the β-Barrel Assembly Machine

The β-barrel assembly machinery (BAM) mediates folding and insertion of β-barrel outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. BAM is a five-protein complex consisting of the β-barrel OMP BamA and lipoproteins BamB, -C, -D, and -E. High resolution structures of all the individual BAM subunits and a BamD-BamC complex have been determined. However, the overall complex architecture remains elusive. BamA is the central component of BAM and consists of a membrane-embedded β-barrel and a periplasmic domain with five polypeptide translocation-associated (POTRA) motifs thought to interact with the accessory lipoproteins. Here we report the crystal structure of a fusion between BamB and a POTRA3–5 fragment of BamA. Extended loops 13 and 17 protruding from one end of the BamB β-propeller contact the face of the POTRA3 β-sheet in BamA. The interface is stabilized by several hydrophobic contacts, a network of hydrogen bonds, and a cation-π interaction between BamA Tyr-255 and BamB Arg-195. Disruption of BamA-BamB binding by BamA Y255A and probing of the interface by disulfide bond cross-linking validate the physiological relevance of the observed interface. Furthermore, the structure is consistent with previously published mutagenesis studies. The periplasmic five-POTRA domain of BamA is flexible in solution due to hinge motions in the POTRA2–3 linker. Modeling BamB in complex with full-length BamA shows BamB binding at the POTRA2–3 hinge, suggesting a role in modulation of BamA flexibility and the conformational changes associated with OMP folding and insertion.     

Biogenesis of CFTR and other Polytopic Membrane Proteins: New Rolesfor the Ribosome-Translocon Complex

Polytopic protein biogenesis represents a critical, yet poorly understood area of modern biology with important implications for human disease. Inherited mutations in a growing array of membrane proteins frequently lead to improper folding and/or trafficking. The cystic fibrosis transmembrane conductance regulator (CFTR) is a primary example in which point mutations disrupt CFTR folding and lead to rapid degradation in the endoplasmic reticulum (ER). It has been difficult, however, to discern the mechanistic principles of such disorders, in part, because membrane protein folding takes place coincident with translation and within a highly specialized environment formed by the ribosome, Sec61 translocon, and the ER membrane. This ribosome-translocon complex (RTC) coordinates the synthesis, folding, orientation and integration of transmembrane segments across and into the ER membrane. At the same time, RTC function is controlled by specific sequence determinants within the nascent polypeptide. Recent studies of CFTR and other native membrane proteins have begun to define novel variations in translocation pathways and to elucidate the specific steps that establish complex topology. This article will attempt to reconcile advances in our understanding of protein biogenesis with emerging models of RTC function. In particular, it will emphasize how information within the nascent polypeptide is interpreted by and in turn controls RTC dynamics to generate the broad structural and functional diversity observed for naturally occurring membrane proteins.Abbreviations: AQP, aquaporin; CFTR, cystic fibrosis transmembrane conductance regulator; ECL, extracellular loop; EM, electron microscopy; ER, endoplasmic reticulum; ICL, intracellular loop; PTC, peptidyltransferase center; RNC, ribosome-nascent chain; RTC, ribosome-translocon complex; SRP, signal recognition particle; SR, SRP receptor; TM, transmembrane (segment); TMD, transmembrane domain. ABC, ATP binding cassette; BiP, heavy chain binding protein; FRET, Förster resonance energy transfer; NBD, nucleotide binding domain; SPC, signal peptidase complex; TrAF, translocation-associated factors; TRAM, translocating chain-associated membrane protein; TRAP, translocon-associated protein.     

Insights into Peroxisome Function from the Structure of PEX3 in Complex with a Soluble Fragment of PEX19

The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.