Anke Edelmann

Assembly of phosphofructokinase-1 from Saccharomyces cerevisiae in extracts of single-deletion mutants

Phosphofructokinase‐1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non‐identical subunits, α and β, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly and reactivation of the enzyme have been studied in cell‐free extracts of single‐deletion mutants. In contrast to the previously described lack of phosphofructokinase‐1 activity in cell‐free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts. This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption.

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, α and β, 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 α‐ or β‐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 α‐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 α‐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. Copyright

A Novel Form of 6-Phosphofructokinase IDENTIFICATION AND FUNCTIONAL RELEVANCE OF A THIRD TYPE OF SUBUNIT IN PICHIA PASTORIS

Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of α subunits and α/β subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, α, β, and γ. The sequence of the γ subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the γ subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the γ subunit is not required for enzymatic activity, the γ subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and γ subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the γ subunit. Therefore, we suggest that the γ subunit contributes to adaptation of P. pastoris to energy resources.

Molecular genetics of 6-phosphofructokinase in Pichia pastoris

Previously, studies on glucose‐induced microautophagy in the methylotrophic yeast Pichia pastoris provided evidence that the glucose‐induced selective autophagy‐1‐protein is the α‐subunit of 6‐phosphofructokinase (Pfk), a key enzyme in the glycolytic pathway. In our work, we could clearly demonstrate that two types of subunits of Pfk exist in P. pastoris. Investigating the yeast cell‐free extract by Western blot analysis, two distinct signals of Pfk were obtained. In addition, we isolated a DNA sequence containing the complete ORF of PpPFK2 encoding the β‐subunit of Pfk from P. pastoris with a deduced molecular mass of 103.7 kDa. On the basis of these results, a hetero‐oligomeric structure of Pfk in P. pastoris became obvious. Because the molecular and kinetic properties of a homo‐oligomeric yeast Pfk appear to be more similar to those of mammalian Pfk, as described in the literature, our results are of interest for the growing number of studies on P. pastoris as a heterologous production system. Furthermore, the 3′‐ and 5′‐non‐coding regions of PpPFK2 were isolated and several putative binding sites for regulatory factors could be identified in the promoter region. The sequence has been deposited in the GenBank data library under Accession No. AF395078. Copyright

C-terminal modification of 6-phosphofructo-1-kinase from Saccharomyces cerevisiae and its influence on enzyme structure and activity.

Studies on limited proteolysis of 6-phosphofructo-1-kinase (Pfk-1) from Saccharomyces cerevisiae led to the suggestion that the C-terminal part of the alpha-subunit must contribute to the stabilisation of the octameric enzyme structure. To analyse the role of the C-terminus in vivo, the respective terminus of one of both types of subunits of Pfk-1 was sequentially truncated or extended. These modifications resulted in a decrease of the protein level of the mutated subunit and of the specific enzyme activity in the cell-free extract as well as in changes of the kinetic properties. Size exclusion HPLC demonstrated that the modified subunit is still able to assemble with the native counterpart generating an enzymatically active hetero-octamer. On the basis of our results we assume that the C-termini are important for the three-dimensional structure of the subunits determining their susceptibility to proteolysis and the ability to assembly to an active, oligomeric Pfk-1.

3D structure of phosphofructokinase from Pichia pastoris: localization of the novel γ-subunits

The largest and one of the most complex ATP-dependent allosteric phosphofructokinase (Pfk) has been found in the methylotrophic yeast, Pichia pastoris. The enzyme is a hetero-oligomer ( approximately 1MDa) composed of three distinct subunits (alpha, beta and gamma) with molecular masses of 109, 104 and 41kDa, respectively. While the alpha- and beta-subunits show sequence similarities to other phosphofructokinase subunits, the gamma-subunit does not show high homology to any known protein in the databases. We have determined the first quaternary structure of P. pastoris phosphofructokinase by 3D electron microscopy. Random conical techniques and tomography have been instrumental to ascertain the quality of the sample preparations for structural studies and to obtain a reliable 3D structure. The final reconstruction of P. pastoris Pfk resembles its yeast counterparts with four additional densities, assigned to four gamma-subunits, bridging the N-terminal domains of the four pairs of alpha- and beta-subunits. Our data has evidenced novel interactions between the gamma- and the alpha-subunits comparable in intensity to the interactions, shown by cross-linking and limited proteolytic degradation experiments, between the gamma- and beta-subunits. The structural data provides clear insights into the allosteric fine-tuned regulation of the enzyme by ATP and AMP observed in this yeast species.