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Published on: June 13, 2021
Fungal ABC transporter deletion and localization analysis
Andriy Kovalchuk1, Stefan S Weber, Jeroen G Nijland
1Faculty of Agriculture and Forestry, Department of Forest Sciences, Forest Pathology, University of Helsinki, Helsinki, Finland. a.kovalchuk@rug.nl
Methods in Molecular Biology (Clifton, N.J.)
|December 21, 2011
Summary
This study details a protocol for analyzing ABC transporter function in Penicillium chrysogenum. It combines gene deletion with GFP-tagging and organelle staining for accurate intracellular localization.
Area of Science:
- Mycology
- Cell Biology
- Biochemistry
Background:
- Fungal metabolism is compartmentalized within membrane-bound organelles.
- Understanding the intracellular localization of transporter proteins is crucial for elucidating their biological functions.
- Green fluorescent protein (GFP) fusion expression is a common method for visualizing protein localization in fungal cells.
Purpose of the Study:
- To provide a detailed protocol for the deletion and localization analysis of ATP-binding cassette (ABC) transporters in Penicillium chrysogenum.
- To establish a cost-efficient method for studying transporter protein function in filamentous fungi.
- To enable the visualization and functional characterization of specific fungal transporters.
Main Methods:
- Construction and transformation of expression plasmids for ABC transporter gene deletion.
- Cultivation of Penicillium chrysogenum transformants.
- Microscopy analysis of protein localization using GFP fusions.
- Staining of fungal cells with organelle-specific dyes (Hoechst 33342, MitoTracker DeepRed, FM4-64) for co-localization studies.
Main Results:
- A comprehensive protocol for genetic manipulation and localization studies of ABC transporters in P. chrysogenum was successfully established.
- The method allows for the visualization of transporter proteins within specific subcellular compartments.
- The combined approach of gene deletion and GFP-tagging facilitates the functional analysis of transporter proteins.
Conclusions:
- The developed protocol is a valuable tool for investigating the roles of ABC transporters in fungal physiology and metabolism.
- Accurate intracellular localization data aids in understanding transporter function and potential drug targets.
- This methodology can be adapted for studying other transporter families in filamentous fungi.
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01:31ABC Transporters: Exporter
ATP-binding cassette or ABC transporter is the largest superfamily of integral membrane proteins. The transporters have transmembrane-binding domains (TMDs) and nucleotide-binding domains (NBDs). The TMDs are specific to their substrates, whereas the NBDs are similar to engines that complete ATP hydrolysis to complete the substrate transport. They can be full transporters consisting of two TMDs and NBDs, half transporters with one TMD and NBD, while some encoded with a single TMD or NBD are...
01:27ABC Transporters: Importer
ATP-binding cassette or ABC transporters are a class of ATP-driven pumps that hydrolyze ATP to move solutes across the membrane. They can be grouped into importers and exporters. While exporters are present in all domains of life, importers exist only in bacteria and some plants.
In bacteria, based on the number of transmembrane helices and the chemical nature of their substrates, the ABC importers can be divided into three types:
In bacteria, based on the number of transmembrane helices and the chemical nature of their substrates, the ABC importers can be divided into three types: