Interaction of 6-phosphofructokinase with cytosolic proteins of Saccharomyces cerevisiae.
Hetero-octameric 6-phosphofructokinase (Pfk-1) from Saccharomyces cerevisiae is composed of two types of subunits, alpha and beta, which are encoded by the unlinked genes PFK1 and PFK2. Pfk single deletion mutants expressing only one type of subunit exhibit Pfk-1 activity in vivo which, however, is completely lost immediately after cell disruption. In order to elucidate the preconditions of the in vivo activity of the mutant enzymes composed of either alpha- or beta-subunits, we have investigated their potential interaction with selected heat shock and cytoskeletal proteins, employing co-immunoprecipitation and immunofluorescence microscopy. Western blot analysis identified the mitochondrial chaperonin Hsp60, as well as the cytoskeleton proteins alpha-tubulin and actin, in complexes with Pfk-1 that were co-precipitated from a cell-free extract of a pfk2 single deletion mutant expressing only the alpha-subunit. The interaction of the corresponding mutant enzyme and Hsp60 was found to depend on the ATP concentration of the extract. Immunofluorescence microscopy displayed a conspicuously filamentous arrangement of the Pfk-1 mutant protein, exclusively in the pfk2 single deletion mutant. The analysis of structure and activity of Pfk-1 expressed in S. cerevisiae mutant strains defective in various heat shock proteins (TRiC/CCT, Hsp70, Hsp 104) and in the respective wild-type background did not reveal significant differences.
Schwock
Jörg
J
Institut für Biochemie der Medizinischen Fakultät der Universität Leipzig, Liebigstrasse 16, D-04103 Leipzig, Germany.
Kirchberger
Jürgen
J
Edelmann
Anke
A
Kriegel
Thomas M
TM
Kopperschläger
Gerhard
G
D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011233:Precipitin Tests; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism
D000199:Actins; D006360:Heat-Shock Proteins; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; D010732:Phosphofructokinase-1