Natalie L. Catlett

Split-Marker Recombination for Efficient Targeted Deletion of Fungal Genes

A commonly used method for fungal gene deletion is introduction of linear DNA consisting of a selectable marker gene flanked on both sides by short stretches of DNA that target a gene of interest (W irsel et al 1996 Curr. Genet 29:241-249). Gene deletion in Cochliobolus heterostrophus and Gibberella zeae occurs efficiently with this approach. To facilitate deletion construct synthesis, we have applied the split-marker” deletion strategy previously developed for Saccharomyces cerevisiae (Fairhead et al. 1996 Y east 12:1439-57; Fairhead et al. 1998 Gene 223:33-46). Here, we describe both fusion PCR-based and plasmid-based deletion methods using this strategy with PEG-mediated protoplast transformation (Turgeon et al, 1985 M ol. Gen. Genet. 201:450-453). These methods are predicted to work well with any transformable fungus that undergoes homologous recombination between chromosomal and introduced DNA sequences.

Whole-Genome Analysis of Two-Component Signal Transduction Genes in Fungal Pathogens

ABSTRACT Two-component phosphorelay systems are minimally comprised of a histidine kinase (HK) component, which autophosphorylates in response to an environmental stimulus, and a response regulator (RR) component, which transmits the signal, resulting in an output such as activation of transcription, or of a mitogen-activated protein kinase cascade. The genomes of the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans encode one, three, and three HKs, respectively. In contrast, the genome sequences of the filamentous ascomycetes Neurospora crassa, Cochliobolus heterostrophus (Bipolaris maydis), Gibberella moniliformis (Fusarium verticillioides), and Botryotinia fuckeliana (Botrytis cinerea) encode an extensive family of two-component signaling proteins. The putative HKs fall into 11 classes. Most of these classes are represented in each filamentous ascomycete species examined. A few of these classes are significantly more prevalent in the fungal pathogens than in the saprobe N. crassa, suggesting that these groups contain paralogs required for virulence. Despite the larger numbers of HKs in filamentous ascomycetes than in yeasts, all of the ascomycetes contain virtually the same downstream histidine phosphotransfer proteins and RR proteins, suggesting extensive cross talk or redundancy among HKs.

Vac7p, a novel vacuolar protein, is required for normal vacuole inheritance and morphology.

During cell division, the vacuole of Saccharomyces cerevisiae partitions between mother and daughter cells. A portion of the parental vacuole membrane moves into the bud, and ultimately membrane scission divides the vacuole into two separate structures. Here we characterize two yeast mutations causing defects in vacuole membrane scission, vac7-1 and vac14-1. A third mutant, afab1-2 strain, isolated in a nonrelated screen (A. Yamamoto et al., Mol. Biol. Cell 6:525-539, 1995) shares the vacuolar phenotypes of the vac7-1 and vac14-1 strains. Unlike the wild type, mutant vacuoles are not multilobed structures; in many cases, a single vacuole spans both the mother and bud, with a distinct gap in the mother-bud neck. Thus, even where the membranes are closely opposed, vacuole fission is arrested. Simply enlarging the vacuole does not produce this mutant phenotype. An additional common phenotype of these mutants is a defect in vacuole acidification; however, vacuole scission in most other vacuole acidification mutants is normal. An alteration in vacuole membrane lipids could account for both the vacuole membrane scission and acidification defects. Because a directed screen has not identified additional class III complementation groups, it is likely that all three genes are involved in a similar process. Interestingly, FAB1, was previously shown to encode a putative phosphatidylinositol-4-phosphate 5-kinase. Moreover, overexpression of FAB1 suppresses the vac14-1 mutation, which suggests that VAC14 and FAB1 act at a common step. VAC7 encodes a novel 128-kDa protein that is localized at the vacuole membrane. This location of Vac7p is consistent with its involvement in vacuole morphology and inheritance.

Regulated degradation of a class V myosin receptor directs movement of the yeast vacuole

Normal cellular function requires that organelles be positioned in specific locations. The direction in which molecular motors move organelles is based in part on the polarity of microtubules and actin filaments. However, this alone does not determine the intracellular destination of organelles. For example, the yeast class V myosin, Myo2p, moves several organelles to distinct locations during the cell cycle. Thus the movement of each type of Myo2p cargo must be regulated uniquely. Here we report a regulatory mechanism that specifically provides directionality to vacuole movement. The vacuole-specific Myo2p receptor, Vac17p, has a key function in this process. Vac17p binds simultaneously to Myo2p and to Vac8p, a vacuolar membrane protein. The transport complex, Myo2p–Vac17p–Vac8p, moves the vacuole to the bud, and is then disrupted through the degradation of Vac17p. The vacuole is ultimately deposited near the centre of the bud. Removal of a PEST sequence (a potential signal for rapid protein degradation) within Vac17p causes its stabilization and the subsequent ‘backward’ movement of vacuoles, which mis-targets them to the neck between the mother cell and the bud. Thus the regulated disruption of this transport complex places the vacuole in its proper location. This may be a general mechanism whereby organelles are deposited at their terminal destination.

Identification of an organelle-specific myosin V receptor

Class V myosins are widely distributed among diverse organisms and move cargo along actin filaments. Some myosin Vs move multiple types of cargo, where the timing of movement and the destinations of selected cargoes are unique. Here, we report the discovery of an organelle-specific myosin V receptor. Vac17p, a novel protein, is a component of the vacuole-specific receptor for Myo2p, a Saccharomyces cerevisiae myosin V. Vac17p interacts with the Myo2p cargo-binding domain, but not with vacuole inheritance-defective myo2 mutants that have single amino acid changes within this region. Moreover, a region of the Myo2p tail required specifically for secretory vesicle transport is neither required for vacuole inheritance nor for Vac17p–Myo2p interactions. Vac17p is localized on the vacuole membrane, and vacuole-associated Myo2p increases in proportion with an increase in Vac17p. Furthermore, Vac17p is not required for movement of other cargo moved by Myo2p. These findings demonstrate that Vac17p is a component of a vacuole-specific receptor for Myo2p. Organelle-specific receptors such as Vac17p provide a mechanism whereby a single type of myosin V can move diverse cargoes to distinct destinations at different times.

Divide and multiply: organelle partitioning in yeast

The mechanisms ensuring accurate partitioning of yeast vacuoles and mitochondria are distinct, yet they share common elements. Both organelles move along actin filaments, and both organelles require fusion and fission to maintain normal morphology. Recent studies have revealed that while vacuolar inheritance requires a processive myosin motor, mitochondrial inheritance requires controlled actin polymerization. Distinct sets of proteins required for the fusion and fission of each organelle have also been identified.

Myosin V attachment to cargo requires the tight association of two functional subdomains

The myosin V carboxyl-terminal globular tail domain is essential for the attachment of myosin V to all known cargoes. Previously, the globular tail was viewed as a single, functional entity. Here, we show that the globular tail of the yeast myosin Va homologue, Myo2p, contains two structural subdomains that have distinct functions, namely, vacuole-specific and secretory vesicle–specific movement. Biochemical and genetic analyses demonstrate that subdomain I tightly associates with subdomain II, and that the interaction does not require additional proteins. Importantly, although neither subdomain alone is functional, simultaneous expression of the separate subdomains produces a functional complex in vivo. Our results suggest a model whereby intramolecular interactions between the globular tail subdomains help to coordinate the transport of multiple distinct cargoes by myosin V.

A Point Mutation in the Cargo-Binding Domain of Myosin V Affects Its Interaction with Multiple Cargoes

ABSTRACT Class V myosins move diverse intracellular cargoes, which attach via interaction of cargo-specific proteins to the myosin V globular tail. The globular tail of the yeast myosin V, Myo2p, contains two structural and functional subdomains. Subdomain I binds to the vacuole-specific protein, Vac17p, while subdomain II likely binds to an as yet unidentified secretory vesicle-specific protein. All functions of Myo2p require the tight association of subdomains I and II, which suggests that binding of a cargo to one subdomain may inhibit cargo-binding to a second subdomain. Thus, two types of mutations are predicted to specifically affect a subset of Myo2p cargoes: first are mutations within a cargo-specific binding region; second are mutations that mimic the inhibited conformation of one of the subdomains. Here we analyze a point mutation in subdomain I, myo2-2(G1248D), which is likely to be this latter type of mutation. myo2-2 has no effect on secretory vesicle movement. The secretory vesicle binding site is in subdomain II. However, myo2-2 is impaired in several Myo2p-related functions. While subdomains I and II of myo2-2p tightly associate, there are measurable differences in the conformation of its globular tail. Based solely on the ability to restore vacuole inheritance, a set of intragenic suppressors of myo2-2 were identified. All suppressor mutations reside in subdomain I. Moreover, subdomain I and II interactions occurred in all suppressors, demonstrating the importance of subdomain I and II association for Myo2p function. Furthermore, 3 of the 10 suppressors globally restored all tested defects in myo2-2. This large proportion of global suppressors strongly suggests that myo2-2(G1248) causes a conformational change in subdomain I that simultaneously affects multiple cargoes.