The AbcA transporter of Staphylococcus aureus affects cell autolysis.

Increased production of penicillin-binding protein PBP 4 is known to increase peptidoglycan cross-linking and contributes to methicillin resistance in Staphylococcus aureus. The pbp4 gene shares a 400-nucleotide intercistronic region with the divergently transcribed abcA gene, encoding an ATP-binding cassette transporter of unknown function. Our study revealed that methicillin stimulated abcA transcription but had no effects on pbp4 transcription. Analysis of abcA expression in mutants defective for global regulators showed that abcA is under the control of agr. Insertional inactivation of abcA by an erythromycin resistance determinant did not influence pbp4 transcription, nor did it alter resistance to methicillin and other cell wall-directed antibiotics. However, abcA mutants showed spontaneous partial lysis on plates containing subinhibitory concentrations of methicillin due to increased spontaneous autolysis. Since the autolytic zymograms of cell extracts were identical in mutants and parental strains, we postulate an indirect role of AbcA in control of autolytic activities and in protection of the cells against methicillin.

Insertion of lysosomal targeting sequences to the amyloid precursor protein reduces secretion of beta A4.

The processing of the amyloid precursor protein (APP) was investigated in cells stably expressing different APP hybrid proteins. The cytoplasmic domain of APP was either deleted or replaced by the corresponding domain of the membrane protein TGN38, lamp-1, or LIMPII. The cytosolic domain of TGN38 in the APP molecule did not alter the secretion of beta A4 when compared with the wild-type APP; however, APP associated with the cell surface and the nonamyloidogenic processing of APP were reduced. With the APP molecules carrying the lysosomal targeting signals of lamp-1 or LIMPII, a decrease in the secretion of beta A4 was observed. Cell surface association and nonamyloidogenic processing were also impaired. This suggests increased degradation of APP and thus efficient targeting to the lysosomal system. Cells expressing the Swedish APP variant generated intracellular beta A4 that accumulated after treatment with chloroquine. This effect was more dramatic with APP mutants carrying lysosomal targeting signals than with full-length APP. Our data suggest the existence of an intracellular site of beta A4 generation from where beta A4 is degraded rather than secreted.

Effect of alkalizing agents on the processing of the beta-amyloid precursor protein.

We investigated the processing pathway of the amyloid precursor protein (APP) to the secretion of beta A4 under the treatment of ammonium chloride (NH4Cl), bafilomycin A1 (bafA1), or chloroquine, all three agents thought to raise the pH in acidic compartments. HEK-293 cells expressing wild-type APP (APPwt) and APP carrying the Swedish double mutation (APPswe) were affected in a different manner: while cells expressing APPswe decreased the secretion of beta A4 after treatment with bafA1 and NH4Cl, cells expressing APPwt compensated the drug-induced decrease in beta A4 by an increased generation of alternative beta A4-related peptides. Within cells APP accumulated, while the formation of a C-terminal fragment of APP generated by beta-secretase was completely inhibited. Thus, BafA1 and NH4Cl reduced the secretion of beta A4 by inhibiting beta-secretase. Treatment with chloroquine did not alter beta A4 secretion but, strikingly, resulted in an accumulation of intracellular beta A4. The effect of reduced APP endocytosis was studied by expressing APP molecules lacking the cytoplasmic domain (APPwt.delta. APPswe.delta). Truncation of APP reduced beta A4 secretion from APPwt but not from APPswe. BafA1 and NH4Cl treatment inhibited the formation of beta A4 in cells expressing APPswe.delta but not APPwt.delta. With these constructs, chloroquine had no effect and no accumulation of intracellular beta A4 was observed. Since alkalizing agents still affected endocytosis-deficient APP containing the Swedish double mutation, we suggest that the formation of beta A4 from this mutated APP takes place mainly in an acidic compartment along the constitutive secretory pathway. Much in contrast to this, beta A4 generation from APPwt appears to occur also in the endosomal/lysosomal compartment.

Intracellular accumulation of beta-amyloid in cells expressing the Swedish mutant amyloid precursor protein.

beta-Amyloid (beta A) is a normal metabolic product of the amyloid precursor protein (APP) that accumulates in senile plaques in Alzheimer's disease. Cells that express the Swedish mutant APP (Sw-APP) associated with early onset Alzheimer's disease overproduce beta A. In this report, we show that expression of Sw-APP gives rise to cell-associated beta A, which is not detected in cells that express wild-type APP. Cell-associated beta A is rapidly generated, is trypsin-resistant, and is not derived from beta A uptake, indicating that it is generated from intracellular processing of Sw-APP. Intracellular and secreted beta A are produced with different kinetics. The generation of intracellular beta A is partially resistant to monensin and a 20 degrees C temperature block but is completely inhibited by brefeldin A, suggesting that it occurs in the Golgi complex. Monensin, brefeldin A, and a 20 degrees C temperature block almost completely inhibit beta A secretion without causing increased cellular retention of beta A, suggesting that secreted beta A is generated in a post-Golgi compartment. These results suggest that the metabolism of Sw-APP gives rise to intracellular and secreted forms of beta A through distinct processing pathways. Pathological conditions may therefore alter both the level and sites of accumulation of beta A. It remains to be determined whether the intracellular form of beta A plays a role in the formation of amyloid plaques.

Organ-specific expression of highly divergent thionin variants that are distinct from the seed-specific crambin in the crucifer Crambe abyssinica.

Most thionins of higher plants are toxic to various bacteria, fungi, and animal and plant cells. The only known exception is the seed-specific thionin, crambin, of the crucifer Crambe abyssinica. Crambin has no net charge, is very hydrophobic and exhibits no toxicity. In the present work, the organization of the crambin precursor polypeptide was deduced from cD-NA sequences. The precursor shows a domain structure similar to that of the preproprotein of other thionins, which contains a signal peptide, a thionin domain and a C-terminal amino acid extension. Unlike the thionin precursors studied thus far, both the thionin domain and the C-terminal amino acid extension of the crambin precursor have no net charge and are hydrophobic, thus facilitating their interaction, by analogy to that proposed for the corresponding domains of other thionin precursors that have positive and negative charges. The existence of a large number of novel and highly variable thionin variants in Crambe abyssinica has been deduced from cDNA sequences that were amplified by the polymerase chain reaction (PCR) from RNA of seeds, leaves and cotyledons. While the deduced amino acid sequences of the thionin domains of most of these thionin precursor molecules are highly divergent, the two other domains are conserved. Most of the predicted thionin variants are positively charged. The presence of positively charged residues in the thionin domains consistently correlates with the presence of a negatively charged residue in the C-terminal amino acid extension of the various thionin precursors. The different thionin variants are encoded by distinct sets of genes and are expressed in an organ-specific manner.

cDNA-derived identification of novel thionin precursors in Viscum album that contain highly divergent thionin domains but conserved signal and acidic polypeptide domains.

The existence of new thionin variants in Viscum album has been deduced from cDNA sequences. Unlike the viscotoxins and related thionins previously found in different members of the Viscaceae, these novel thionins contain eight rather than six cysteine residues. In this respect they resemble thionins described previously from various cereals and from Pyrularia pubera, which also contain eight cysteine residues at identical positions. All of the new thionins of V. album are encoded as higher-molecular-weight precursors consisting of a signal peptide, a thionin domain and an acidic polypeptide domain. While the deduced amino acid sequences of the thionin domains of different precursor molecules are highly divergent, the two other domains are conserved among all of the variants and are distinct from the corresponding domains of thionin precursors of other plant species.

The Anticyclic Timing of Leaf Senescence in the Parasitic Plant Viscum album Is Closely Correlated with the Selective Degradation of Sulfur-Rich Viscotoxins.

Leaf senescence and abscission have been studied in the semi-parasitic plant mistletoe (Viscum album). Leaf senescence and abscission occur in the summer, when the metabolic activity of the host has reached its maximum. In contrast with their hosts, mistletoes selectively degrade only one major leaf protein during leaf senescence, the sulfur-rich viscotoxin, whereas most of the remaining leaf proteins are lost during abscission. The changes in viscotoxin content are paralleled by changes in the concentration of the corresponding mRNA. Shortly before the onset of leaf senescence, the mRNA for viscotoxin has disappeared from the leaves. The anticyclic timing of leaf senescence and the degradation of only one major leaf protein seems to reflect an adaptation of the parasite to its habitat.