PMID:35536002

[manulera]

PMID:35536002

Activities and Structure-Function Analysis of Fission Yeast Inositol Pyrophosphate (IPP) Kinase-Pyrophosphatase Asp1 and Its Impact on Regulation of pho1 Gene Expression

Inositol pyrophosphates (IPPs) are signaling molecules that regulate cellular phosphate homeostasis in diverse eukaryal taxa. In fission yeast, mutations that increase 1,5-IP8 derepress the PHO regulon while mutations that ablate IP8 synthesis are PHO hyper-repressive. Fission yeast Asp1, the principal agent of 1,5-IP8 dynamics, is a bifunctional enzyme composed of an N-terminal IPP kinase domain and a C-terminal IPP pyrophosphatase domain. Here we conducted a biochemical characterization and mutational analysis of the autonomous Asp1 kinase domain (aa 1-385). Reaction of Asp1 kinase with IP6 and ATP resulted in both IP6 phosphorylation to 1-IP7 and hydrolysis of the ATP γ-phosphate, with near-equal partitioning between productive 1-IP7 synthesis and unproductive ATP hydrolysis under optimal kinase conditions. By contrast, reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus ATP hydrolysis. Alanine scanning identified essential constituents of the active site. We deployed the Ala mutants to show that derepression of pho1 expression correlated with Asp1's kinase activity. In the case of full-length Asp1, the activity of the C-terminal pyrophosphatase domain stifled net phosphorylation of the 1-position during reaction of Asp1 with ATP and either IP6 or 5-IP7. We report that inorganic phosphate is a concentration-dependent enabler of net IP8 synthesis by full-length Asp1 in vitro, by virtue of its antagonism of IP8 turnover. IMPORTANCE Expression of the fission yeast phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate (IPP) signaling molecule 1,5-IP8. IP8 dynamics are determined by Asp1, a bifunctional enzyme comprising N-terminal IPP 1-kinase and C-terminal IPP 1-pyrophosphatase domains that catalyze IP8 synthesis and catabolism, respectively. Here, we interrogated the activities and specificities of the Asp1 kinase domain and full length Asp1. We find that reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus the significant unproductive ATP hydrolysis seen during its reaction with IP6. We report that full-length Asp1 catalyzes futile cycles of 1-phosphate phosphorylation by its kinase component and 1-pyrophosphate hydrolysis by its pyrophosphatase component that result in unproductive net consumption of the ATP substrate. Net synthesis of 1,5-IP8 is enabled by physiological concentrations of inorganic phosphate that selectively antagonize IP8 turnover.

Intro

IP7 and IP8 are singaling molecules involved in eukaryal phosphate homeostasis -> transcriptional response to phosphate availability, upregulates genes involved in extracellular phosphate acquisition.

IP6 -Kcs1-> 5-IP7 -Asp1-> 1,5-IP8

• Asp1 is a bifunctional enzyme:

  • N-term IPP kinase: forward reaction
  • C-term pyrophosphatase:reverse reaction

• Isolated N-term has the activity

• Fission yeast phosphate homeostasis (PHO) regulon three phosphate acquisition genes:

  • [x] pho1 (cell surface acid phosphatase)
  • [x] pho84: (phosphate membrane transporter)
  • [x] tgp1: (glycerophosphate transporter)

• [x] Reressed by flancking lncRNAs prt, prt2 and nc-tgp1

  • By transcribing, they displace the Pho7 transcription factor from DNA binding sites

• asp1 that cannot do pirophospatase reaction (asp1-H397A) increases IP8 levels and derepresses PHO regulon -> termination of prt lncRNA synthesis.

• asp1 deletion (no IP8) -> pho1 hyperrepression

• [x] asp1 syntehtic lethal with CPF subunits mutations: maybe IP8 or IP7 important for 3'-processing / termination events (agonist of pol2 transc termination)

Main conclusions:

• N-term kinase domain (1-385):
  • IP6 -> IP7 + ATP hydrolysis
  • Asp1 can hydrolise ATP even without substrate (and ratio between use of substrate and simple ATP hydrolysis depends on Mg2+ concentration)
  • Asp1 is 22x faster on IP7 phosphorylation than on IP6, favours that instead of ATP hydrolysis.
• Full prot:
  • No Ip6 to IP7 due to pyrophosphatase activity

Results

Fig. 1

• [x] GO annotation extensionresidues(1-385)
• Asp1(1-385):
  • ~IP6 -> IP7~
  • [x] ATP hydrolisis with or without IP6
• ~Asp1(1-385)(D333A) -> neither activity. They do the mutation on the trunacted one (see KDa in Fig. 1C).~ Already annotated to D333A

Fig. 3

• ~The enzyme uses ATP as a nucleotide~ Implied in the GO term

Fig. 4

• Asp1(1-385) cannot phosphorylate 1-IP&, only 5-IP7. Indicated in the GO term.
• Asp1(1-385) favours kinase activity vs ATPase when substrate is 5-IP7. CAnnot capture

Fig. 7

• Mutations on Asp1(1-385)
• [x] eliminated IP7 kinase activity or reduced product formation to less than 5% of wild type (H204A, R223A, K260A, D321A, and D333A)
• [x] did not affect IP7 kinase activity (R285A and K341A)
• [x] modestly reduced activity vis-à-vis wild type: R123A (82% of WT), R293A (64%), N335A (45%), K43A (30%), and K224A (18%)

Fig. 8

• Cells that are asp1D and express asp1(1-385) mutants from a plasmid, tgp1 promoter, and plasmid bears a nc-tgp1 lncRNA under nmt1 promoter.

  • No thiamine -> lncRNA is expressed -> suppression of tgp1 promoter expression of asp1 mutant
  • Thiamine -> lncRNA not expressed -> active tgp1 promoter expresses asp1 mutants.

• The assay measures the Pho1 acid phosphatase activity in cells:

  • [x] Constitutively expressing the wt N-term -> high acid phosphatase activity (pressumably pho1 gene expression is induced)
  • [x] In the wt -> activity is 5.7 vs. 157
  • [x] This is lower in mutants to alanine, lowest in those that inactivated the enzyme in vitro
  • [x] R123A lower levels

Fig. 10

• Full-length enzyme: Asp1 converted 1-IP7 to IP6 but did not modify either the 5-IP7 or IP6 substrates (Fig. 10C), thereby affirming previous findings that Asp1 is specific for hydrolysis of the phosphoanhydride bond at the 1-pyrophosphate position. Already captured in GO
• [X] Asp1-H397A pyrophosphatase mutant pyrophosphatase mutant.

Fig. 12

• Constitutive expression of full-length Asp1 increased the Pho1 activity
  • Higher even if the pyrophosphatase is repressed.

Summary:

strong preference of Asp1 kinase for 5-IP7 versus IP6

FYPO

• [x] NTR: abolished inositol diphosphatase activity
• [x] NTR: normal inositol hexakisphosphate kinase activity

GO

Existing annotations.

• [ ] pyrophosphatase-defective asp1-H397A refs 9,10. This has to be changed:
  • abolished inositol 1-diphosphate 2,3,4,5,6-pentakisphosphate 1-diphosphatase activity, in https://curation.pombase.org/pombe/curs/893c5c2ae65e3990
• [X] GO:0052846 and GO:0101012 are probably the same thing

Ask the authors:

Discuss with Val

The experiment quantifies the acid phosphatase activity activity, pressumably of Pho1 (regulated by the PHO regulon). Mutations in asp1 enzyme can lead to the activation/repression of pho1 expression, and therefore high/low enzymatic activity. The authors have annotated:

• increased termination of RNA polymerase II transcription
• abnormal regulation of transcription by nutrient

However, this is not really what was measured, but rather the pho1 activity. At the same time, I am not sure a molecular function phenotype is correct either, since these phenotypes are probably to indicate that enzymatic activity is altered at the molecule level, not because there is more / less enzyme.

I guess in this case what makes more sense is to use "increased / decreased protein level" for pho1?

[manulera]

Hi @ValWood a question for you for this paper. See the secion "Discuss with Val"

[manulera]

Hi @ValWood ccing here again im case you didnt see. No hurry for this

[ValWood]

Yes sorry I had scanned and not spotted the specific question.

Exactly. I used increased protein level (inferred from Enzyme assay data). in the other paper https://curation.pombase.org/pombe/curs/fcfd9a50a6a6cb53 (bottom).