In Gram-negative bacteria, outer-membrane integrity is essential for survival and is monitored by the σE stress-response system, which initiates damage-repair pathways. One activating signal is unassembled outer-membrane proteins. Using biochemical and genetic experiments in Escherichia coli, we found that off-pathway intermediates in lipopolysaccharide transport and assembly provided an additional required signal. These distinct signals, arising from disruptions in the transport and assembly of the major outer-membrane components, jointly determined the rate of proteolytic destruction of a negative regulator of the σE transcription factor, thereby modulating expression of stress-response genes. This dual-signal system permits a rapid response to dysfunction in outer-membrane biogenesis, while buffering responses to transient fluctuations in individual components, and may represent a broad strategy for bacteria to monitor their interface with the environment.
Photosystem II (PSII) catalyzes the first of two photosynthetic reactions that convert sunlight into chemical energy. Native PSII is a supercomplex consisting of core and light-harvesting chlorophyll proteins. Although the structure of PSII has been resolved by x-ray crystallography, the mechanism underlying its assembly is poorly understood. Here, we report that an immunophilin of the chloroplast thylakoid lumen is required for accumulation of the PSII supercomplex in Arabidopsis thaliana. The immunophilin, FKBP20-2, belongs to the FK-506 binding protein (FKBP) subfamily that functions as peptidyl-prolyl isomerases (PPIases) in protein folding. FKBP20-2 has a unique pair of cysteines at the C terminus and was found to be reduced by thioredoxin (Trx) (itself reduced by NADPH by means of NADP-Trx reductase). The FKBP20-2 protein, which contains only two of the five amino acids required for catalysis, showed a low level of PPIase activity that was unaffected on reduction by Trx. Genetic disruption of the FKBP20-2 gene resulted in reduced plant growth, consistent with the observed lower rate of PSII activity determined by fluorescence (using leaves) and oxygen evolution (using isolated chloroplasts). Analysis of isolated thylakoid membranes with blue native gels and immunoblots showed that accumulation of the PSII supercomplex was compromised in mutant plants, whereas the levels of monomer and dimer building blocks were elevated compared with WT. The results provide evidence that FKBP20-2 participates specifically in the accumulation of the PSII supercomplex in the chloroplast thylakoid lumen by means of a mechanism that has yet to be determined.chloroplast thylakoid lumen ͉ protein folding ͉ photosynthetic electron transport M uch of life on Earth is sustained by oxygenic photosynthesis, a process that utilizes sunlight to produce oxygen and organic carbon from water and carbon dioxide. The absorption of light and its conversion into chemical energy is brought about by two photosystems [photosystem II (PSII) and photosystem I (PSI)] acting sequentially. PSII catalyzes the lightdependent oxidation of water that results in the evolution of oxygen. The electrons released in this reaction are transferred along a photosynthetic electron transport chain that leads, by means of PSI, to the production of NADPH and ATP, the chemical energy currency used for carbon fixation.The chloroplast PSII core complex consists of Ϸ17 protein subunits that include the D1 and D2 reaction centers for chlorophyll (Chl) P680 binding, cytochrome b559, CP43 and CP47 for building the Chl antennae, and other proteins whose function is less well characterized (1-4). The native form of PSII residing in the thylakoid membrane is believed to be a supercomplex consisting of the core and peripheral light-harvesting complex II (LHCII) components. The light harvested by LHCII is transferred to the core complex that brings about charge separation, thereby driving the transfer of electrons from water to plastoquinone and initiating photosynthetic electr...
SummaryA novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a baculovirus vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and eotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this eosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH 2 terminus of an MCP is the biologically relevant species.T he number of chemokines of the CC subfamily has grown considerably during the past few years, and important new information has been gathered about their activities on different types of leukocytes (1). Of particular interest were the findings that the monocyte chemotactic proteins (MCPs) are not only effective on monocytes (2-4), but also attract CD4 + and CD8 + T lymphocytes (5, 6) and basophil leukocytes (7-10). MCP-3 (10) and R_ANTES (11) were also shown to be powerful attractants of eosinophil leukocytes and, most recently, eotaxin, a CC chemokine with marked sequence similarity to MCP-3, was found to share this activity and to be unusually selective for eosinophils (12-14).In a program aiming at the discovery of human genes by large-scale sequencing of partial cDNA clones, a novel chemokine cDNA was identified in a library constructed from human fetal mRNA. The full-length cDNA was cloned into a baculovirus expression vector, and the chemokine obtained was designated CK[310. A screening for the induction of changes of the cytosolic free Ca 2+ concentration ([Ca2+]i) in human blood monocytes showed that CK[310 was as effective as MCP-1 and MCP-3. We compared its biological activity on human monocytes, neutrophils, eosinophils, and T lymphocytes with that ofMCP-1, MCP-2, MCP-3, RANTES, MIP-lc~, and eotaxin, and found that CK[310 is functionally very similar to MCP-3. Since the novel CC chemokine also shares marked sequence identity to the monocyte chemotactic proteins, we adopted the term MCP-4. This designation is actually not new. A 153-bp PCR fragment was previously cloned from h...
The balance between cholesterol and sphingolipids within the plasma membrane has long been implicated in endocytic membrane trafficking. However, in contrast to cholesterol functions, little is still known about the roles of sphingolipids and their metabolites. Perturbing the cholesterol/sphingomyelin balance was shown to induce narrow tubular plasma membrane invaginations enriched with sphingosine kinase 1 (SphK1), the enzyme that converts the bioactive sphingolipid metabolite sphingosine to sphingosine-1-phosphate, and suggested a role for sphingosine phosphorylation in endocytic membrane trafficking. Here we show that sphingosine and sphingosine-like SphK1 inhibitors induced rapid and massive formation of vesicles in diverse cell types that accumulated as dilated late endosomes. However, much smaller vesicles were formed in SphK1-deficient cells. Moreover, inhibition or deletion of SphK1 prolonged the lifetime of sphingosine-induced vesicles. Perturbing the plasma membrane cholesterol/sphingomyelin balance abrogated vesicle formation. This massive endosomal influx was accompanied by dramatic recruitment of the intracellular SphK1 and Bin/Amphiphysin/Rvs domain-containing proteins endophilin-A2 and endophilin-B1 to enlarged endosomes and formation of highly dynamic filamentous networks containing endophilin-B1 and SphK1. Together, our results highlight the importance of sphingosine and its conversion to sphingosine-1-phosphate by SphK1 in endocytic membrane trafficking.
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