Abstract. The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in protein translocation across both the outer and inner mitochondrial membranes was studied using two temperature-sensitive yeast mutants. The degree of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twice by the processing peptidase. A short aminoterminal segment of the preprotein (40-60 amino acids) was driven into the matrix by the membrane potential, independent of hsp70 function, allowing a single cleavage of the presequence. Artificial unfolding of the preprotein allowed complete translocation into the matrix in the case where mutant mt-hsp70 had detectable binding activity. However, in the mutant mitochondria in which binding to mt-hsp70 could not be detected the mature part of the preprotein was only translocated to the intermembrane space. We propose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins. (a) Mt-hsp70 facilitates unfolding of the polypeptide chain for translocation across the mitochondrial membranes. (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the completion of transport of a matrix-targeted preprotein across the inner membrane. This second role is independent of the folding state of the preprotein, thus identifying mt-hsp70 as a genuine component of the inner membrane translocation machinery. Furthermore we determined the sites of the mutations and show that both a functional ATPase domain and ATP are needed for mt-hsp70 to bind to the polypeptide chain and drive its translocation into the matrix.

SummaryPseudomonas aeruginosa is an opportunistic pathogen and a leading cause of hospital-acquired pneumonia. We identified a 73 kDa protein, designated Pseudomonas exoprotein A (PepA), that was secreted by P. aeruginosa strain PA103. PepA was necessary for in vitro killing of epithelial cells as well as virulence in a mouse model of acute pneumonia. Several properties of PepA suggested that it was secreted by a type III system. Secretion occurred without cleavage of a signal peptide and in low-calcium environments in the presence of a divalent cation chelator, as is the case for characterized P. aeruginosa type III secreted proteins. Secretion of PepA was absent from isogenic mutants with defective type III pathways. Finally, amino-terminal peptide sequence analysis indicated that the amino-terminal five residues of PepA were identical to those of ExoS and ExoT, two type III secreted proteins of P. aeruginosa. After secretion, PepA underwent cleavage at two sites, each with the sequence A-X-K-S, suggesting that the cleavage may be caused by a protease. The gene encoding PepA, designated pepA, was cloned and sequenced, and comparisons with the genetic database using BLAST alignments indicated that the nucleotide sequence of pepA and the inferred protein sequence of PepA had no homology to known sequences. A nucleotide sequence identical to the consensus element for binding of ExsA, a transcriptional activator of P. aeruginosa type III secretion genes, was located 84 bp 5Ј of the translational start codon. Analysis of transposon insertion mutants indicated that the carboxy terminus was required for cytotoxicity. Examination of respiratory clinical isolates demonstrated that pepA was a variable trait and probably acquired by horizontal transmission. Consistent with this hypothesis was the identification of a putative insertion element 94 bp 5Ј of the PepA translational start site. Analysis of G þ C content of the PepA coding sequence and the adjacent insertion element suggested that they were acquired together from a different species. In summary, PepA is a secreted protein of P. aeruginosa that is necessary for epithelial cell cytotoxicity in vitro and virulence in a mouse model of pneumonia.

We report the isolation and characterization of a previously unidentified Escherichia coli gene that suppresses the temperature-sensitive growth and filamentation of a dnaK deletion mutant strain. Introduction of a multicopy plasmid carrying this wild-type gene into a dnaK deletion mutant strain rescued the temperature-sensitive growth of the dnaK deletion mutant strain at 40.5 degrees C and the filamentation, fully at 37 degrees C and partially at 40.5 degrees C. However, the inability of dnaK mutant cells to support bacteriophage lambda growth was not suppressed. This gene was also able to suppress the temperature-sensitive growth of a grpE280 mutant strain at 41 degrees C. Filamentation of the grpE280 mutant strain was suppressed at 37 degrees C but not at 41 degrees C. The dnaK suppressor gene, designated dksA, maps near the mrcB gene (3.7 min on the E. coli chromosome). DNA sequence analysis and in vivo experiments showed that dksA encodes a 17,500-Mr polypeptide. Gene disruption experiments indicated that dksA is not an essential gene.

In both animal and yeast cells, signaling pathways involving small guanosine triphosphatases (GTPases) regulate polarized organization of the actin cytoskeleton. In the budding yeast Saccharomyces cerevisiae, the Ras-like GTPase Bud1/Rsr1 and its guanosine 5'-diphosphate (GDP)/guanosine 5'-triphosphate (GTP) exchange factor Bud5 are involved in the selection of a specific site for growth, thus determining cell polarity. We found that Bud5 is localized at the cell division site and the presumptive bud site.

SummaryVirulence of Pseudomonas aeruginosa involves the co-ordinate expression of a range of factors including type IV pili (tfp), the type III secretion system (TTSS) and quorum sensing. Tfp are required for twitching motility, efficient biofilm formation, and for adhesion and type III secretion (TTS)-mediated damage to mammalian cells. We describe a novel gene ( fimL ) that is required for tfp biogenesis and function, for TTS and for normal biofilm development in P. aeruginosa . The predicted product of fimL is homologous to the Nterminal domain of ChpA, except that its putative histidine and threonine phosphotransfer sites have been replaced with glutamine. fimL mutants resemble vfr mutants in many aspects including increased autolysis, reduced levels of surface-assembled tfp and diminished production of type III secreted effectors. Expression of vfr in trans can complement fimL mutants. vfr transcription and production is reduced in fimL mutants whereas cAMP levels are unaffected. Deletion and insertion mutants of fimL frequently revert to wild-type phenotypes suggesting that an extragenic suppressor mutation is able to overcome the loss of fimL . vfr transcription and production, as well as cAMP levels, are elevated in these revertants, while Pseudomonas quinolone signal (PQS) production is reduced. These results suggest that the site(s) of spontaneous mutation is in a gene(s) which lies upstream of vfr transcription, cAMP, production, and PQS synthesis. Our studies indicate that Vfr and FimL are components of intersecting pathways that control twitching motility, TTSS and autolysis in P. aeruginosa .

Biphasic activation of Cdc42 by Bud3 and then Cdc24 during G1 of the yeast cell cycle is necessary for assembly of a proper bud site.

In the budding yeast Saccharomyces cerevisiae, selection of the bud site determines the axis of polarized cell growth and eventual oriented cell division. Bud sites are selected in specific patterns depending on cell type. These patterns appear to depend on distinct types of marker proteins in the cell cortex; in particular, the bipolar budding of diploid cells depends on persistent landmarks at the birth-scar-distal and -proximal poles that involve the proteins Bud8p and Bud9p, respectively. Rax1p and Rax2p also appear to function specifically in bipolar budding, and we report here a further characterization of these proteins and of their interactions with Bud8p and Bud9p. Rax1p and Rax2p both appear to be integral membrane proteins. Although commonly used programs predict different topologies for Rax2p, glycosylation studies indicate that it has a type I orientation, with its long N-terminal domain in the extracytoplasmic space. Analysis of rax1 and rax2 mutant budding patterns indicates that both proteins are involved in selecting bud sites at both the distal and proximal poles of daughter cells as well as near previously used division sites on mother cells. Consistent with this, GFP-tagged Rax1p and Rax2p were both observed at the distal pole as well as at the division site on both mother and daughter cells; localization to the division sites was persistent through multiple cell cycles. Localization of Rax1p and Rax2p was interdependent, and biochemical studies showed that these proteins could be copurified from yeast. Bud8p and Bud9p could also be copurified with Rax1p, and localization studies provided further evidence of interactions. Localization of Rax1p and Rax2p to the bud tip and distal pole depended on Bud8p, and normal localization of Bud8p was partially dependent on Rax1p and Rax2p. Although localization of Rax1p and Rax2p to the division site did not appear to depend on Bud9p, normal localization of Bud9p appeared largely or entirely dependent on Rax1p and Rax2p. Taken together, the results indicate that Rax1p and Rax2p interact closely with each other and with Bud8p and Bud9p in the establishment and/or maintenance of the cortical landmarks for bipolar budding.

Yeast cells organize their actin cytoskeleton in a highly polarized manner during vegetative growth. The Ras-like GTPase Rsr1/Bud1 and its regulators are required for selection of a specific site for growth. Here we showed that Rsr1/Bud1 was broadly distributed on the plasma membrane and highly concentrated at the incipient bud site and polarized growth sites. We also showed that localization of Cdc24, a guanine nucleotide exchange factor for the Cdc42 GTPase, to the proper bud site was dependent on Rsr1/Bud1. Surprisingly, Rsr1/ Bud1 also localized to intracellular membranes. A mutation in the lysine repeat in the hypervariable region of Rsr1/Bud1 specifically abolished its plasma membrane localization, whereas a mutation at the CAAX motif eliminated both plasma membrane and internal membrane association of Rsr1/Bud1. Thus the lysine repeat and the CAAX motif of Rsr1/Bud1 are important for its localization to the plasma membrane and to the polarized growth sites. This localization of Rsr1/Bud1 is essential for its function in proper bud site selection because both mutations resulted in random bud site selection.Cells of the budding yeast Saccharomyces cerevisiae undergo oriented cell division by selecting a specific site for polarized growth on their cell cortex (1-3). Haploid a and ␣ cells bud in an axial pattern, whereas diploid a/␣ cells bud in a bipolar pattern. The GTPase module consisting of Rsr1/Bud1 (Rsr1 hereafter), its GDP-GTP exchange factor Bud5, and its GTPase-activating protein Bud2 is essential for selecting the proper site for polarized growth in both haploid and diploid cells (4 -7). Based on genetic and biochemical data, we proposed previously that the Rsr1 GTPase module directs bud site assembly to occur at specific locations by recruiting components such as Cdc24 required for bud formation to that site (8). Recruiting these proteins to the presumptive bud site is thought to direct the cytoskeleton and secretory apparatus toward the bud site, thereby restricting new growth to the bud (9). One of the key questions in understanding the molecular basis of cell polarity is how specific sites for actin polymerization are determined.To understand the mechanism of action of the Rsr1 GTPase module, it is crucial to determine whether any of its components are localized to the presumptive bud site. We reported previously that both Bud2 and Bud5 are localized to the presumptive bud site and to discrete sites during the cell cycle (10, 11). This localization is essential for selection of a specific site for growth. Here we report that Rsr1 is broadly distributed on the plasma membrane and is highly concentrated at the incipient bud site and polarized growth sites. Mutational studies indicated that the lysine repeat in the hypervariable region and the CAAX motif of Rsr1 are important for its localization and its role in selection of a proper site for growth. We also show that localization of Cdc24, a guanine nucleotide exchange factor for Cdc42, to the proper bud site is dependent on Rsr1. EXPERIMENTAL PR...