Light-dependent degradation of the Q(B)-protein in isolated pea thylakoids

The 32 000-dalton Q_B-protein of photosystem II (PS II) is rapidly damaged and removed from isolated pea thylakoids during incubation in the light resulting in a loss of photosynthetic electron flow through PS II. This in vitro photoinhibition is similar to that previously reported with intact Chlamydomonas cells. The damage occurs at a faster rate in vitro, however, due to the inability of isolated thylakoids to synthesize replacement Q_B-protein. The removal of the damaged Q_B-protein does not require any soluble components of the chloroplast stroma and is unaffected by the protease inhibitors phenyl-methylsulfonylfluoride or antipain. Unlike the effect of trypsin, no low mol. wt. membrane-bound or soluble fragments of the labelled Q_B-protein could be identified either by autoradiography or immunologically using polyclonal antibodies specific for the Q_B-protein. The lightinduced damage to the Q_B-protein (indicated by a loss of Q_B functional activity), preceded the removal of the protein from the membrane. We conclude that photodamage of the Q_B-protein generates a conformational change which renders the protein susceptible to attack by a highly efficient, intrinsic membrane protease.     

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)⁺RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45°C induces the expression of ˜10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)⁺RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36°C to 42°C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)⁺RNAs endure in the cells at 42°C up to 5 h but are no longer translated in vivo, whereas some poly(A)⁻RNAs persist and are translated. As in pea, a poly(A)⁺RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)⁺RNA coding for the 22-kd HSP appears after 1 h at 36°C. Its synthesis increases with the temperature of incubation up to 42°C, although it decreases after ˜2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26°C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.     

CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.     

Correlation between changes in the membrane organization and susceptibility to phospholipase C attack induced by ATP depletion of rat erythrocytes

About 20 and 43% of the total membrane phospholipids are hydrolized in fresh rat erythrocytes by treatment with phospholipase C (Bacillus cereus), or both sphingomyelinase and phospholipase C, respectively, without causing cell lysis. Treatment of ATP-depleted cells with phospholipase C alone results in 50% hydrolysis and extensive lysis. Depletion of ATP causes a marked increase in the aggregation of intramembranous particles accompanied by a similar increase in the smooth area between the particle clusters as revealed by the freeze-etch technique. Such changes are not induced by extensive phospholipid hydrolysis in absence of cell lysis in fresh cells.Based on these and additional data, it is suggested that the membrane phospholipid organization can be divided into 3 types: phospholipids exposed to phospholipase C; phospholipids protected against phospholipase C by presence of sphingomyelin; phospholipids which can be exposed following alteration of the proteinlipid interactions. Such alterations which might be induced by a variety of means, including ATP depletion, might result in clustering of intramembranous particles and increase of the free lipid bilayer phase of the membrane.     

Computational analyses reveal spatial relationships between nuclear pore complexes and specific lamins

Nuclear lamin isoforms form fibrous meshworks associated with nuclear pore complexes (NPCs). Using datasets prepared from subpixel and segmentation analyses of 3D–structured illumination microscopy images of WT and lamin isoform knockout mouse embryo fibroblasts, we determined with high precision the spatial association of NPCs with specific lamin isoform fibers. These relationships are retained in the enlarged lamin meshworks of Lmna^−/− and Lmnb1^−/− fibroblast nuclei. Cryo-ET observations reveal that the lamin filaments composing the fibers contact the nucleoplasmic ring of NPCs. Knockdown of the ring-associated nucleoporin ELYS induces NPC clusters that exclude lamin A/C fibers but include LB1 and LB2 fibers. Knockdown of the nucleoporin TPR or NUP153 alters the arrangement of lamin fibers and NPCs. Evidence that the number of NPCs is regulated by specific lamin isoforms is presented. Overall the results demonstrate that lamin isoforms and nucleoporins act together to maintain the normal organization of lamin meshworks and NPCs within the nuclear envelope.     

Virulence Gene Profiling and Pathogenicity Characterization of Non-Typhoidal Salmonella Accounted for Invasive Disease in Humans

Human infection with non-typhoidal Salmonella serovars (NTS) infrequently causes invasive systemic disease and bacteremia. To understand better the nature of invasive NTS (iNTS), we studied the gene content and the pathogenicity of bacteremic strains from twelve serovars (Typhimurium, Enteritidis, Choleraesuis, Dublin, Virchow, Newport, Bredeney, Heidelberg, Montevideo, Schwarzengrund, 9,12:l,v:- and Hadar). Comparative genomic hybridization using a Salmonella enterica microarray revealed a core of 3233 genes present in all of the iNTS strains, which include the Salmonella pathogenicity islands 1–5, 9, 13, 14; five fimbrial operons (bcf, csg, stb, sth, sti); three colonization factors (misL, bapA, sinH); and the invasion gene, pagN. In the iNTS variable genome, we identified 16 novel genomic islets; various NTS virulence factors; and six typhoid-associated virulence genes (tcfA, cdtB, hlyE, taiA, STY1413, STY1360), displaying a wider distribution among NTS than was previously known. Characterization of the bacteremic strains in C3H/HeN mice showed clear differences in disease manifestation. Previously unreported characterization of serovars Schwarzengrund, 9,12:l,v:-, Bredeney and Virchow in the mouse model showed low ability to elicit systemic disease, but a profound and elongated shedding of serovars Schwarzengrund and 9,12:l,v:- (as well as Enteritidis and Heidelberg) due to chronic infection of the mouse. Phenotypic comparison in macrophages and epithelial cell lines demonstrated a remarkable intra-serovar variation, but also showed that S. Typhimurium bacteremic strains tend to present lower intracellular growth than gastroenteritis isolates. Collectively, our data demonstrated a common core of virulence genes, which might be required for invasive salmonellosis, but also an impressive degree of genetic and phenotypic heterogeneity, highlighting that bacteremia is a complex phenotype, which cannot be attributed merely to an enhanced invasion or intracellular growth of a particular strain.