Related Experiment Video

Updated: Jan 30, 2026

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents
09:42

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

Published on: August 7, 2013

10.8K

Hyperactive mutation occurs adjacent to the essential glutamate 286 for transport in the yeast tryptophan permease

Kaori Amano1, Ryoga Ishii1, Takahiro Mochizuki1

  • 1Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, Sagamihara, Japan.

Biochemical and Biophysical Research Communications
|January 21, 2019
PubMed

Insights

Hyperactive mutations in the Tat2 permease enhance tryptophan import in yeast, enabling growth at very low tryptophan concentrations. These findings offer insights into high-affinity amino acid transport mechanisms.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Yeast Genetics

Background:

  • High-affinity tryptophan import in Saccharomyces cerevisiae is primarily mediated by the plasma membrane permease Tat2.
  • Understanding the mechanisms of high-affinity amino acid transport is crucial for various biological processes.

Purpose of the Study:

  • To identify and characterize mutations in Tat2 that enhance tryptophan import efficiency.
  • To investigate the impact of these mutations on Tat2's kinetic properties and cellular localization.
  • To elucidate the structural and functional basis for improved high-affinity tryptophan transport.

Main Methods:

  • Site-directed mutagenesis to create Tat2 variants (I285V, I285T).
  • Yeast growth assays at varying tryptophan concentrations.
  • Kinetic analysis (Km, Vmax) of tryptophan uptake.
  • Western blotting and fluorescence microscopy to assess Tat2 protein levels and localization.

Main Results:

  • Identified hyperactive Tat2 mutations (I285V, I285T) enabling yeast growth at <4 μg/mL tryptophan.
  • Tat2I285V and Tat2I285T exhibited significantly lower Km values (17 and 11 μg/mL, respectively) compared to wild-type Tat2 (24 μg/mL).
  • Mutations resulted in a 2-fold increase in Vmax/Km, indicating enhanced affinity and transport efficiency.
  • Tat2I285V and Tat2I285T showed rapid increases in cellular levels and efficient cell surface localization under low tryptophan conditions.

Conclusions:

  • The I285V and I285T mutations enhance Tat2's affinity for tryptophan, likely by altering the local residue environment and facilitating proton symport.
  • These hyperactive permease variants offer a valuable tool for studying high-affinity transport.
  • The findings provide a strategy for improving the functionality of various amino acid permeases.
Keywords:
Hyperactive mutationSaccharomyces cerevisiaeTryptophan permease Tat2

More Related Videos

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET12:07

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

Published on: October 9, 2021

3.8K
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia05:51

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

Published on: June 15, 2011

26.4K

Related Concept Videos

Mutations01:39

Mutations

Overview
94.5K
Viral Mutations00:36

Viral Mutations

A mutation is a change in the sequence of bases of DNA or RNA in a genome. Some mutations occur during replication of the genome due to errors made by the polymerase enzymes that replicate DNA or RNA. Unlike DNA polymerase, RNA polymerase is prone to errors because it is not capable of “proofreading” its work. Viruses with RNA-based genomes, like HIV, therefore accrue mutations faster than viruses with DNA-based genomes. Because mutation and recombination provide the raw material...
39.9K
Regulation of Expression Occurs at Multiple Steps02:24

Regulation of Expression Occurs at Multiple Steps

Gene expression can be regulated at almost every step from gene to protein. Transcription is the step that is most commonly regulated. This involves the binding of proteins to short regulatory sequences on the DNA. This association can either promote or inhibit the transcription of a gene associated with the respective sequence.
Transcription results in the generation of precursor (pre-mRNA) that consists of both exons and introns, which needs further processing before being translated to a...
26.4K
Regulation of Expression Occurs at Multiple Steps02:24

Regulation of Expression Occurs at Multiple Steps

4.0K
Yeast Signaling01:28

Yeast Signaling

Yeasts are single-celled organisms, but unlike bacteria, they are eukaryotes (cells with a nucleus). Cell signaling in yeast is similar to signaling in other eukaryotic cells. A ligand, such as a protein or a small molecule released from a yeast cell, attaches to a receptor on the cell surface. The binding stimulates second-messenger kinases to activate or inactivate transcription factors that further regulate gene expression. Many of the yeast intracellular signaling cascades have similar...
17.3K
Facilitated Transport01:19

Facilitated Transport

The chemical and physical properties of plasma membranes cause them to be selectively permeable. Since plasma membranes have both hydrophobic and hydrophilic regions, substances need to be able to transverse both regions. The hydrophobic area of membranes repels substances such as charged ions. Therefore, such substances need special membrane proteins to cross a membrane successfully. In  facilitated transport, also known as facilitated diffusion, molecules and ions travel across a...
147.9K