The molecular architecture of the yeast plasma membrane H+-ATPase phosphorylation region was explored by Fe2+-catalyzed cleavage. An ATP-Mg2+·Fe2+ complex was found to act as an affinity cleavage reagent in the presence of dithiothreitol/H2O2. Selective enzyme cleavage required bound adenine nucleotide, either ATP or ADP, in the presence of Mg2+. The fragment profile included a predominant N-terminal 61-kDa fragment, a minor 37-kDa fragment, and three prominent C-terminal fragments of 39, 36, and 30 kDa. The 61-kDa N-terminal and 39-kDa C-terminal fragments were predicted to originate from cleavage within the conserved MLT558GDAVG sequence. The 37-kDa fragment was consistent with cleavage within the S4/M4 sequence PVGLPA340V, while the 30-kDa and 36-kDa C-terminal fragments appeared to originate from cleavage in or around sequences D646TGIAVE and DMPGS595 ELADF, respectively. The latter are spatially close to the highly conserved motif GD634GVND638APSL and conserved residues Thr558 and Lys615, which have been implicated in coordinating Mg2+ and ATP. Overall, these results demonstrate that Fe2+ associated with ATP and Mg2+ acts as an affinity cleavage agent of the H+-ATPase with back-bone cleavage occurring in conserved regions known to coordinate metal-nucleotide complexes. This study provides support for a three-dimensional organization of the phosphorylation region of the yeast plasma membrane H+-ATPase that is consistent with, but not identical to, typical P-type enzymes.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology