Reference: Ma J, et al. (2008) Localization of autophagy-related proteins in yeast using a versatile plasmid-based resource of fluorescent protein fusions. Autophagy 4(6):792-800

Reference Help

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.

Reference Type
Evaluation Studies | Journal Article | Research Support, Non-U.S. Gov't
Authors
Ma J, Bharucha N, Dobry CJ, Frisch RL, Lawson S, Kumar A
Primary Lit For
Additional Lit For
Review For

Interaction Annotations


Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details about experiment type and any other genes involved in the interaction.

Interactor Interactor Type Assay Annotation Action Modification Phenotype Source Reference

Gene Ontology Annotations


Increase the total number of rows showing on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table.

Gene Gene Ontology Term Qualifier Aspect Method Evidence Source Assigned On Annotation Extension Reference

Phenotype Annotations


Increase the total number of rows showing on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details.

Gene Phenotype Experiment Type Mutant Information Strain Background Chemical Details Reference

Regulation Annotations


Increase the total number of rows displayed on this page using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; to filter the table by a specific experiment type, type a keyword into the Filter box (for example, “microarray”); download this table as a .txt file using the Download button or click Analyze to further view and analyze the list of target genes using GO Term Finder, GO Slim Mapper, SPELL, or YeastMine.

Regulator Target Experiment Assay Construct Conditions Strain Background Reference