[Show abstract][Hide abstract] ABSTRACT: During the budding of COPII vesicles from transitional ER (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover, and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain (CCD), which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.
Full-text · Article · Sep 2013 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: The morphogenetic transition between yeast and filamentous forms of the human fungal pathogen Candida albicans is regulated by a variety of signaling pathways. How these pathways interact to orchestrate morphogenesis, however, has not been as well characterized. To address this question and to identify genes that interact with the Regulation of Ace2 and Morphogenesis (RAM) pathway during filamentation, we report the first large-scale genetic interaction screen in C. albicans. Our strategy for this screen was based on the concept of complex haploinsufficiency (CHI). A heterozygous mutant of CBK1
(cbk1Δ/CBK1), a key RAM pathway protein kinase, was subjected to transposon-mediated, insertional mutagenesis. The resulting double heterozygous mutants (6,528 independent strains) were screened for decreased filamentation on Spider Medium (SM). From the 441 mutants showing altered filamentation, 139 transposon insertion sites were sequenced, yielding 41 unique CBK1-interacting genes. This gene set was enriched in transcriptional targets of Ace2 and, strikingly, the cAMP-dependent protein kinase A (PKA) pathway, suggesting an interaction between these two pathways. Further analysis indicates that the RAM and PKA pathways co-regulate a common set of genes during morphogenesis and that hyper-activation of the PKA pathway may compensate for loss of RAM pathway function. Our data also indicate that the PKA–regulated transcription factor Efg1 primarily localizes to yeast phase cells while the RAM–pathway regulated transcription factor Ace2 localizes to daughter nuclei of filamentous cells, suggesting that Efg1 and Ace2 regulate a common set of genes at separate stages of morphogenesis. Taken together, our observations indicate that CHI–based screening is a useful approach to genetic interaction analysis in C. albicans and support a model in which these two pathways regulate a common set of genes at different stages of filamentation.
[Show abstract][Hide abstract] ABSTRACT: Transposon mutagenesis is an effective method for generating large sets of random mutations in target DNA, with applicability toward numerous types of genetic screens in prokaryotes, single-celled eukaryotes, and metazoans alike. Relative to methods of random mutagenesis by chemical/UV treatment, transposon insertions can be easily identified in mutants with phenotypes of interest. The construction of transposon insertion mutants is also less labor-intensive on a genome-wide scale than methods for targeted gene replacement, although transposon insertions are not precisely targeted to a specific residue, and thus coverage of the target DNA can be problematic. The collective advantages of transposon mutagenesis have been well demonstrated in studies of the budding yeast Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans, as transposon mutagenesis has been used extensively for phenotypic screens in both yeasts. Consequently, we present here protocols for the generation and utilization of transposon-insertion DNA libraries in S. cerevisiae and C. albicans. Specifically, we present methods for the large-scale introduction of transposon insertion alleles in a desired strain of S. cerevisiae. Methods are also presented for transposon mutagenesis of C. albicans, encompassing both the construction of the plasmid-based transposon-mutagenized DNA library and its introduction into a desired strain of Candida. In total, these methods provide the necessary information to implement transposon mutagenesis in yeast, enabling the construction of large sets of identifiable gene disruption mutations, with particular utility for phenotypic screening in nonstandard genetic backgrounds.
Full-text · Article · Jan 2011 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Protein localization is tightly linked with function, such that the subcellular distribution of a protein serves as an important control point regulating activity. Exploiting this regulatory mechanism, we present here a general approach by which protein location, and hence function, may be controlled on demand in the budding yeast. In this system a small molecule, rapamycin, is used to temporarily recruit a strong cellular address signal to the target protein, placing subcellular localization under control of the selective chemical stimulus. The kinetics of this system are rapid: rapamycin-directed nucleo-cytoplasmic transport is evident 10-12 min post-treatment and the process is reversible upon removal of rapamycin. Accordingly, we envision this platform as a promising approach for the systematic construction of conditional loss-of-function mutants. As proof of principle, we used this system to direct nuclear export of the essential heat shock transcription factor Hsf1p, thereby mimicking the cell-cycle arrest phenotype of an hsf1 temperature-sensitive mutant. Our drug-induced localization platform also provides a method by which protein localization can be uncoupled from endogenous cell signalling events, addressing the necessity or sufficiency of a given localization shift for a particular cell process. To illustrate, we directed the nuclear import of the calcineurin-dependent transcription factor Crz1p in the absence of native stimuli; this analysis directly substantiates that nuclear translocation of this protein is insufficient for its transcriptional activity. In total, this technology represents a powerful method for the generation of conditional alleles and directed mislocalization studies in yeast, with potential applicability on a genome-wide scale.
[Show abstract][Hide abstract] ABSTRACT: The subcellular distribution of kinases and other signaling proteins is regulated in response to cellular cues; however, the extent of this regulation has not been investigated for any gene set in any organism. Here, we present a systematic analysis of protein kinases in the budding yeast, screening for differential localization during filamentous growth. Filamentous growth is an important stress response involving mitogen-activated protein kinase and cAMP-dependent protein kinase signaling modules, wherein yeast cells form interconnected and elongated chains. Because standard strains of yeast are nonfilamentous, we constructed a unique set of 125 kinase-yellow fluorescent protein chimeras in the filamentous Sigma1278b strain for this study. In total, we identified six cytoplasmic kinases (Bcy1p, Fus3p, Ksp1p, Kss1p, Sks1p, and Tpk2p) that localize predominantly to the nucleus during filamentous growth. These kinases form part of an interdependent, localization-based regulatory network: deletion of each individual kinase, or loss of kinase activity, disrupts the nuclear translocation of at least two other kinases. In particular, this study highlights a previously unknown function for the kinase Ksp1p, indicating the essentiality of its nuclear translocation during yeast filamentous growth. Thus, the localization of Ksp1p and the other kinases identified here is tightly controlled during filamentous growth, representing an overlooked regulatory component of this stress response.
Full-text · Article · Aug 2008 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: Plasmid-based collections of fluorescent protein fusions are valuable and versatile resources, facilitating systematic studies of protein localization in multiple genetic backgrounds. At present, however, few such collections exist for the analysis of protein localization in any organism. To address this deficiency, we present here a plasmid-based set of resources for the analysis of protein localization in the budding yeast. Specifically, we constructed a suite of low-copy destination vectors for recombination-based cloning of yeast genes as fluorescent protein fusions. We cloned a set of 384 yeast genes encoding kinases, transcription factors and signaling proteins as "recombination-ready" cassettes; by Gateway cloning, these genes with native promoters can be easily introduced into the destination vectors described above, generating carboxy-terminal fusions to fluorescent proteins. Using these reagents, we constructed a subcollection of 276 genes encoding carboxy-terminal fusions to yellow fluorescent protein (vYFP). This collection encompasses 14 autophagy-related (ATG) genes, and we localized these Atgp-vYFP chimeras during rapamycin-induced autophagy. To illustrate further the utility of this collection as a tool in exploring the functions and interactions of proteins in a pathway, we localized a subset of these Atg-vYFP chimeras in a strain deleted for the scaffolding protein Atg11p. In addition, we validated previous results identifying the integral membrane protein Atg9p at the pre-autophagosomal structure upon overexpression of ATG11 and upon deletion of ATG1. Collectively, this plasmid-based resource of yeast gene-vYFP fusions provides an initial toolkit for a variety of systematic and large-scale localization studies exploring pathway biology in the budding yeast.
[Show abstract][Hide abstract] ABSTRACT: The budding yeast Saccharomyces cerevisiae is well recognized as a preferred eukaryote for the development of genomic technologies and approaches. Accordingly, a sizeable complement of genomic resources has been developed in yeast, and this genomic foundation is now informing a wide variety of disciplines. In particular, yeast genomic methodologies are gaining an expanding foothold in drug development studies, most notably as a preliminary tool towards drug target identification. In this review, we highlight many applications of yeast genomics in the identification of targeted genes and pathways of small molecules or therapeutic drugs. The applicability of genome-wide resources of yeast disruption and deletion mutants for drug-sensitivity/resistance screening is presented here, along with a summary of microarray technologies for drug-based transcriptional profiling and synthetic interaction mapping. Applications of protein-interaction traps for potential drug target identification are also considered. Collectively, this overview of yeast genomics emphasizes the growing intersection between high-throughput model organism biology and medicinal chemistry an intersection promising tangible advances for both academic and pharmaceutical fields alike.
Full-text · Article · Oct 2007 · Combinatorial Chemistry & High Throughput Screening