An NH2-terminal deleted plasma membrane H+-ATPase is a dominant negative mutant and is sequestered in endoplasmic reticulum derived structures.
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.
Masuda
C A
CA
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Centro de Ciencias da Saúde, Universidade Federal do Rio de Janeiro, RJ, Brazil.
Montero-Lomelí
M
M
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000648:ultrastructure; D017384:Sequence Deletion; D014357:Trypsin / Q000378:metabolism
D011994:Recombinant Proteins; D014357:Trypsin; D006180:Proton-Translocating ATPases