Related Experiment Videos
An NH2-terminal deleted plasma membrane H+-ATPase is a dominant negative mutant and is sequestered in endoplasmic
1Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Centro de Ciencias da Saúde, Universidade Federal do Rio de Janeiro, RJ, Brazil.
Abstract:
The NH2-terminus of the plasma membrane H+-ATPase is one of the least conserved segments of this protein among fungi. We constructed and expressed a mutant H+-ATPase from Saccharomyces cerevisiae deleted at an internal peptide within the cytoplasmic NH2-terminus (D44-F116). When the enzyme was subjected to limited trypsinolysis it was digested more rapidly than wild type H+-ATPase. Membrane fractionation experiments and immunofluorescence microscopy, using antibodies against H+-ATPase showed that the mutant ATPase is retained in the endoplasmic reticulum. The pattern observed in the immunofluorescence microscopy resembled structures similar to Russell bodies (modifications of the endoplasmic reticulum membranes) recently described in yeast. When the wild type H+-ATPase was co-expressed with the mutant, wild type H+-ATPase was also retained in the endoplasmic reticulum. Co-expression of both ATPases in a wild type yeast strain was lethal, demonstrating that this is a dominant negative mutant.
Insights
A mutant plasma membrane H+-ATPase lacking an internal NH2-terminal peptide is rapidly digested and retained in the endoplasmic reticulum. Co-expression with wild-type H+-ATPase causes lethality, indicating a dominant negative effect.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Genetics
Background:
- The NH2-terminus of the plasma membrane H+-ATPase is a poorly conserved region among fungi.
- Understanding the function of this region is crucial for comprehending H+-ATPase regulation and localization.
Purpose of the Study:
- To investigate the role of a specific internal peptide within the cytoplasmic NH2-terminus of Saccharomyces cerevisiae H+-ATPase.
- To characterize the functional and localization consequences of deleting this peptide.
Main Methods:
- Construction and expression of a mutant H+-ATPase lacking residues D44-F116.
- Limited trypsinolysis to assess enzyme stability.
- Membrane fractionation and immunofluorescence microscopy to determine protein localization.
- Co-expression studies with wild-type H+-ATPase.
Main Results:
- The mutant H+-ATPase exhibited increased susceptibility to trypsin digestion compared to the wild-type enzyme.
- Immunofluorescence microscopy revealed that the mutant ATPase is retained within the endoplasmic reticulum.
- Co-expression of the mutant with wild-type H+-ATPase led to the retention of both in the endoplasmic reticulum.
- Co-expression of both ATPases in yeast was lethal, confirming a dominant negative phenotype.
Conclusions:
- The deleted NH2-terminal peptide is essential for the proper folding, stability, and/or trafficking of the H+-ATPase.
- Retention in the endoplasmic reticulum suggests a defect in protein maturation or transport.
- The mutant acts as a dominant negative inhibitor, interfering with the function of the wild-type H+-ATPase.