Lois M. Banta

Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases

Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups. Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained. Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B. Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects. In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells. Normal protein secretion appeared to be unaffected in the vpt mutants. The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis. In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes. Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants. However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase. Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants. Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature sensitive for vegetative cell growth. This temperature-sensitive phenotype has been shown to be recessive and to cosegregate with the vacuolar protein-sorting defect in each case. Tetrad analysis showed that vpt3 mapped to the right arm of chromosome XV and that vpt15 mapped to the right arm of chromosome II. Intercrosses with other mutants that exhibited defects in vacuolar protein sorting or function (vpl, sec, pep, and end mutants) revealed several overlaps among these different sets of genes. Together, these data indicate that more than 50 gene products are involved, directly or indirectly, in the process of vacuolar protein sorting.

Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p.

In a late‐Golgi compartment of the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y (CPY) are actively sorted away from the secretory pathway and transported to the vacuole via a pre‐vacuolar, endosome‐like intermediate. The vacuolar protein sorting (vps) mutant vps4 accumulates vacuolar, endocytic and late‐Golgi markers in an aberrant multilamellar pre‐vacuolar compartment. The VPS4 gene has been cloned and found to encode a 48 kDa protein which belongs to the protein family of AAA‐type ATPases. The Vps4 protein was purified and shown to exhibit an N‐ethylmaleimide‐sensitive ATPase activity. A single amino acid change within the AAA motif of Vps4p yielded a protein that lacked ATPase activity and did not complement the protein sorting or morphological defects of the vps4Δ1 mutant. Indeed, when expressed at normal levels in wild‐type cells, the mutant vps4 gene acted as a dominant‐negative allele. The phenotypic characterization of a temperature‐sensitive vps4 allele showed that the immediate consequence of loss of Vps4p function is a defect in vacuolar protein delivery. In this mutant, precursor CPY was not secreted but instead accumulated in an intracellular compartment, presumably the pre‐vacuolar endosome. Electron microscopy revealed that upon temperature shift, exaggerated stacks of curved cisternal membranes (aberrant endosome) also accumulated in the vps4ts mutant. Based on these and other observations, we propose that Vps4p function is required for efficient transport out of the pre‐vacuolar endosome.

Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information

An inactive precursor form of proteinase A (PrA) transits through the early secretory pathway before final vacuolar delivery. We used gene fusions between the gene coding for PrA (PEP4) and the gene coding for the secretory enzyme invertase (SUC2) to identify vacuolar protein-sorting information in the PrA precursor. We found that the 76-amino-acid preprosegment of PrA contains at least two sorting signals: an amino-terminal signal peptide that is cleaved from the protein at the level of the endoplasmic reticulum followed by the prosegment which functions as a vacuolar protein-sorting signal. PrA-invertase hybrid proteins that carried this sequence information were accurately sorted to the yeast vacuole as determined by cell fractionation and immunolocalization studies. Hybrid proteins lacking all or a portion of the PrA prosegment were secreted from the cell. Our gene fusion data together with an analysis of the wild-type PrA protein indicated that N-linked carbohydrate modifications are not required for vacuolar sorting of this protein. Furthermore, results obtained with a set of deletion mutations constructed in the PrA prosegment indicated that this sequence also contributes to proper folding of this polypeptide into a stable transit-competent molecule.

Genome Sequences of Three Agrobacterium Biovars Help Elucidate the Evolution of Multichromosome Genomes in Bacteria

The family Rhizobiaceae contains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, many Rhizobium and Sinorhizobium strains are nitrogen-fixing plant mutualists, while many strains designated as Agrobacterium are plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of the Rhizobiaceae also have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes. Agrobacterium strains have been divided into three biovars, based on physiological and biochemical properties. The genome of a biovar I strain, A. tumefaciens C58, has been previously sequenced. In this study, the genomes of the biovar II strain A. radiobacter K84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the biovar III strain A. vitis S4, a narrow-host-range strain that infects grapes and invokes a hypersensitive response on nonhost plants, were fully sequenced and annotated. Comparison with other sequenced members of the Alphaproteobacteria provides new data on the evolution of multipartite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in select Beta- and Gammaproteobacteria species. Together, these results define the evolution of chromosome architecture and gene content among the Rhizobiaceae and support a generalized mechanism for second-chromosome formation among bacteria.

Genetic and environmental factors affecting T-pilin export and T-pilus biogenesis in relation to flagellation of Agrobacterium tumefaciens.

The T pilus, primarily composed of cyclic T-pilin subunits, is essential for the transmission of the Ti-plasmid T-DNA from Agrobacterium tumefaciens to plant cells. Although the virB2 gene of the 11-gene virB operon was previously demonstrated to encode the full-length propilin, and other genes of this operon have been implicated as members of a conserved transmembrane transport apparatus, the role of each virB gene in T-pilin synthesis and transport and T-pilus biogenesis remained undefined. In the present study, each virB gene was examined and was found to be unessential for T-pilin biosynthesis, except virB2, but was determined to be essential for the export of the T-pilin subunits and for T-pilus formation. We also find that the genes of the virD operon are neither involved in T-pilin export nor T-pilus formation. Critical analysis of three different virD4 mutants also showed that they are not involved in T-pilus biogenesis irrespective of the A. tumefaciens strains used. With respect to the environmental effects on T-pilus biogenesis, we find that T pili are produced both on agar and in liquid culture and are produced at one end of the A. tumefaciens rod-shaped cell in a polar manner. We also report a novel phenomenon whereby flagellum production is shut down under conditions which turn on T-pilus formation. These conditions are the usual induction with acetosyringone at pH 5.5 of Ti-plasmid vir genes. A search of the vir genes involved in controlling this biphasic reaction in induced A. tumefaciens cells revealed that virA on the Ti plasmid is involved and that neither virB nor virD genes are needed for this reaction. The biphasic reaction therefore appears to be mediated through a two-component signal transducing system likely involving an unidentified vir gene in A. tumefaciens.

The plant signal salicylic acid shuts down expression of the vir regulon and activates quormone-quenching genes in Agrobacterium

Agrobacterium tumefaciens is capable of transferring and integrating an oncogenic T-DNA (transferred DNA) from its tumor-inducing (Ti) plasmid into dicotyledonous plants. This transfer requires that the virulence genes (vir regulon) be induced by plant signals such as acetosyringone in an acidic environment. Salicylic acid (SA) is a key signal molecule in regulating plant defense against pathogens. However, how SA influences Agrobacterium and its interactions with plants is poorly understood. Here we show that SA can directly shut down the expression of the vir regulon. SA specifically inhibited the expression of the Agrobacterium virA/G two-component regulatory system that tightly controls the expression of the vir regulon including the repABC operon on the Ti plasmid. We provide evidence suggesting that SA attenuates the function of the VirA kinase domain. Independent of its effect on the vir regulon, SA up-regulated the attKLM operon, which functions in degrading the bacterial quormone N-acylhomoserine lactone. Plants defective in SA accumulation were more susceptible to Agrobacterium infection, whereas plants overproducing SA were relatively recalcitrant to tumor formation. Our results illustrate that SA, besides its well known function in regulating plant defense, can also interfere directly with several aspects of the Agrobacterium infection process.

Altered Profile of Secondary Metabolites in the Root Exudates of Arabidopsis ATP-Binding Cassette Transporter Mutants

Following recent indirect evidence suggesting a role for ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transporter genes highly expressed in the root cells most likely to be involved in secretion processes. Of these 25 genes, we also selected six full-length ABC transporters and a half-size transporter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of these seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals missing in various ABC transporter mutants compared to the wild type when the samples were analyzed by high-performance liquid chromatography-mass spectrometry. These data suggest that more than one ABC transporter can be involved in the secretion of a given phytochemical and that a transporter can be involved in the secretion of more than one secondary metabolite. The primary and secondary metabolites present in the root exudates of the mutants were also analyzed by gas chromatography-mass spectrometry, which allowed for the identification of groups of compounds differentially found in some of the mutants compared to the wild type. For instance, the mutant Atpdr6 secreted a lower level of organic acids and Atmrp2 secreted a higher level of amino acids as compared to the wild type. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters.

Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis.

vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.

Agrobacterium ParA/MinD-like VirC1 spatially coordinates early conjugative DNA transfer reactions

Agrobacterium tumefaciens translocates T‐DNA through a polar VirB/D4 type IV secretion (T4S) system. VirC1, a factor required for efficient T‐DNA transfer, bears a deviant Walker A and other sequence motifs characteristic of ParA and MinD ATPases. Here, we show that VirC1 promotes conjugative T‐DNA transfer by stimulating generation of multiple copies per cell of the T‐DNA substrate (T‐complex) through pairwise interactions with the processing factors VirD2 relaxase, VirC2, and VirD1. VirC1 also associates with the polar membrane and recruits T‐complexes to cell poles, the site of VirB/D4 T4S machine assembly. VirC1 Walker A mutations abrogate T‐complex generation and polar recruitment, whereas the native protein recruits T‐complexes to cell poles independently of other polar processing factors (VirC2, VirD1) or T4S components (VirD4 substrate receptor, VirB channel subunits). We propose that A. tumefaciens has appropriated a progenitor ParA/MinD‐like ATPase to promote conjugative DNA transfer by: (i) nucleating relaxosome assembly at oriT‐like T‐DNA border sequences and (ii) spatially positioning the transfer intermediate at the cell pole to coordinate substrate—T4S channel docking.

The chvH Locus of Agrobacterium Encodes a Homologue of an Elongation Factor Involved in Protein Synthesis

The virulence of Agrobacterium tumefaciens depends on both chromosome- and Ti plasmid-encoded gene products. In this study, we characterize a chromosomal locus, chvH, previously identified by TnphoA mutagenesis and shown to be required for tumor formation. Through DNA sequencing and comparison of the sequence with identified sequences in the database, we show that this locus encodes a protein similar in sequence to elongation factor P, a protein thought to be involved in peptide bond synthesis in Escherichia coli. The analysis of vir-lacZ and vir-phoA translational fusions as well as Western immunoblotting revealed that the expression of Vir proteins such as VirE2 was significantly reduced in the chvH mutant compared with the wild-type strain. The E. coli efp gene complemented detergent sensitivity, virulence, and expression of VirE2 in the chvH mutant, suggesting that chvH and efp are functionally homologous. As expected, ChvH exerts its activity at the posttranscriptional level. Southern analysis suggests that the gene encoding this elongation factor is present as a single copy in A. tumefaciens. We constructed a chvH deletion mutant in which a 445-bp fragment within its coding sequence was deleted and replaced with an omega fragment. On complex medium, this mutant grew more slowly than the wild-type strain, indicating that elongation factor P is important but not essential for the growth of Agrobacterium.