Role of transmembrane segment M8 in the biogenesis and function of yeast plasma-membrane H(+)-ATPase
Related Experiment Video
Updated: Jul 14, 2026
Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle
Published on: March 21, 2025
Guadalupe Guerra1, Valery V Petrov, Kenneth E Allen
1Departmento de Microbiologia, Escuela Nacional de Ciencias Biologicas, I.P.N., Mexico, D.F., Mexico.
Abstract:
Of the four transmembrane helices (M4, M5, M6, and M8) that pack together to form the ion-binding sites of P(2)-type ATPases, M8 has until now received the least attention. The present study has used alanine-scanning mutagenesis to map structure-function relationships throughout M8 of the yeast plasma-membrane H(+)-ATPase. Mutant forms of the ATPase were expressed in secretory vesicles and at the plasma membrane for measurements of ATP hydrolysis and ATP-dependent H(+) pumping. In secretory vesicles, Ala substitutions at a cluster of four positions near the extracytoplasmic end of M8 led to partial uncoupling of H(+) transport from ATP hydrolysis, while substitution of Ser-800 (close to the middle of M8) by Ala increased the apparent stoichiometry of H(+) transport. A similar increase has previously been reported following the substitution of Glu-803 by Gln (Petrov, V. et al., J. Biol. Chem. 275:15709-15718, 2000) at a position known to contribute directly to Ca(2+) binding in the Ca(2+)-ATPase of sarcoplasmic reticulum (Toyoshima, C., et al., Nature 405: 647-655, 2000). Four other mutations in M8 interfered with H(+)-ATPase folding and trafficking to the plasma membrane; based on homology modeling, they occupy positions that appear important for the proper bundling of M8 with M5, M6, M7, and M10. Taken together, these results point to a key role for M8 in the biogenesis, stability, and physiological functioning of the H(+)-ATPase.
Insights
Transmembrane helix M8 is crucial for yeast plasma-membrane H(+)-ATPase function. Mutations in M8 affect ion transport, ATP hydrolysis coupling, and protein stability, revealing its key role in enzyme biogenesis and activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Protein Structure and Function
Background:
- P(2)-type ATPases are vital ion pumps.
- Transmembrane helix M8, part of the ion-binding site, is understudied.
- Understanding M8's role is key to P(2)-type ATPase function.
Purpose of the Study:
- To investigate the structure-function relationships of transmembrane helix M8 in the yeast plasma-membrane H(+)-ATPase.
- To elucidate M8's contribution to ion transport, ATP hydrolysis, and protein stability.
Main Methods:
- Alanine-scanning mutagenesis was employed to create mutant H(+)-ATPases.
- Mutant proteins were expressed in secretory vesicles and at the plasma membrane.
- Measurements of ATP hydrolysis and ATP-dependent H(+) pumping were performed.
Main Results:
- Mutations near the extracytoplasmic end of M8 partially uncoupled H(+) transport from ATP hydrolysis.
- Substitution of Ser-800 with Ala increased the apparent stoichiometry of H(+) transport.
- Other M8 mutations impaired protein folding and trafficking, suggesting roles in helix bundling.
Conclusions:
- Transmembrane helix M8 plays a critical role in yeast H(+)-ATPase biogenesis and stability.
- M8 is essential for the proper physiological functioning of the H(+)-ATPase.
- These findings highlight M8's significance in P(2)-type ATPase mechanisms.
Related Concept Videos
01:19Translocation of Proteins into the Mitochondria
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
01:34Protein Transport into the Inner Mitochondrial Membrane
Transport of mitochondrial precursors across the TIM23 channel is driven by...
01:28Protein Translocation Machinery on the ER Membrane
Sec61 protein conducting channel
In eukaryotes, the translocon complex comprises a core heterotrimeric translocator channel called the Sec61 complex. This channel includes three transmembrane proteins, Sec61α, Sec61β, and Sec61γ, and is the largest subunit of the translocon complex.
01:29Insertion of Multi-pass Transmembrane Proteins in the RER
The multipass transmembrane proteins are the type IV integral membrane proteins with multiple topogenic sequences determining their spatial arrangement in the ER membrane. Nearly all multipass proteins lack a cleavable signal sequence and use...
01:19Membrane Asymmetry Regulating Transporters
Flippase
Eukaryotic flippases are type-IV P-type ATPases or P4-ATPases belonging to P-type ATPase family proteins that are membrane-bound pumps involved in the ATP-mediated transport of ions and molecules across the membrane. Flippases flip specific phospholipids from the outer to the inner leaflet of a membrane. All P4-ATPases have one...
01:37Energy to Drive Translocation
Generally, polypeptides are unfolded by two distinct...