A quantitative screen for metabolic enzyme structures reveals patterns of assembly across the yeast metabolic network

Abstract

Despite the proliferation of proteins that can form filaments or phase-separated condensates, it remains unclear how this behavior is distributed over biological networks. We have found that 60 of the 440 yeast metabolic enzymes robustly form structures, including 10 that assemble within mitochondria. Additionally, the ability to assemble is enriched at branch points on several metabolic pathways. The assembly of enzymes at the first branch point in de novo purine biosynthesis is coordinated, hierarchical, and based on their position within the pathway, while the enzymes at the second branch point are recruited to RNA stress granules. Consistent with distinct classes of structures being deployed at different control points in a pathway, we find that the first enzyme in the pathway, PRPP synthetase, forms evolutionarily conserved filaments that are sequestered in the nucleus in higher eukaryotes. These findings provide a roadmap for identifying additional conserved features of metabolic regulation by condensates/filaments.

FIGURE 1:

Screen of metabolic enzymes reveals 20 new proteins capable of assembly into foci or filaments. (A) Schematic for screening the yeast GFP collection to identify all metabolic enzymes with the ability to form structures. Each strain was grown to log phase, postdiauxic shift, and stationary phase in YPD and fixed in 4% formaldehyde at room temperature for 15 min. Cells were washed and resuspended in 1 M sorbitol before imaging. (B) Multiple growth conditions expand the list of metabolic enzymes forming assemblies. Representative images of metabolic enzymes capable of assembly into filaments or foci in more than 10% of cells. Enzyme names highlighted in yellow represent previously unknown metabolic enzymes forming assemblies. Images were taken from the culture condition with the highest degree of assembly.

FIGURE 2:

Metabolic enzymes can form discrete structures inside and/or outside of the mitochondria. (A) MitoTracker staining reveals differential distribution of metabolic enzyme structures inside or outside the mitochondria. Cells expressing GFP-tagged metabolic enzymes were incubated with 0.1 μM MitoTracker Red for 30 min. Cells were washed and then imaged immediately. (B) Assemblies of metabolic enzymes do not overlap with high-density regions of mitochondria. Cells expressing GFP-tagged metabolic enzymes were transformed with a plasmid containing a dsRed fluorescent protein attached to a mitochondrial targeting sequence (pVTU-mito-dsRED). Dual fluorescent strains were grown to either log phase (Ald4p) or postdiauxic shift (Fum1p, Ilv1p, Ilv2p) and imaged for colocalization.

FIGURE 3:

Enzymes in the de novo purine biosynthetic pathway assemble with different kinetics. (A) Schematic of the de novo purine biosynthetic pathway with yeast orthologues in blue on the left and mammalian orthologues in green on the right. Abbreviations for intermediate metabolites and catalytic enzymes: R5P = ribose-5-phosphate; PRPP = 5-phosphoribosylpyrophosphate; PRA = 5-phosphoribosylamine; GAR = 5-phosphoribosylglycineamide; FGAR = 5′-phosphoribosyl- N-formylglycinamide; FGAM = 5′-phosphoribosyl-N-formylglycinamidine; AIR = 5′-phosphoribosyl-5-aminoimidazole; CAIR = 5′- phosphoribosyl-4-carboxy-5-aminoimidazole; SAICAR = 5′-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole; AICAR = 5-amino-4-imidazolecarboxamide ribotide; FAICAR = 5-formamido-1-(5-phosphoribosyl)-imidazole-4-carboxamide; IMP = inosine-5′-monophosphate; XMP = xanthosine-5′-phosphate; GMP = guanosine-5′-phosphate; GDP = guanosine-5′-diphosphate; SAMP = adenylosuccinate; AMP = adenosine-5′-phosphate; ADP = adenosine-5′-diphosphate; Prs1-5p = phosphoribosylpyrophosphate synthetase; Ade4p = amidophosphoribosyltransferase; Ade5,7p = GAR synthetase/AIR synthetase; Ade8p = GAR transformylase; Ade6p = FGAM synthetase; Ade2p = AIR carboxylase; Ade1p = SAICAR synthase; Ade16/17p = IMP cyclohydrolase; Ade12p = adenylosuccinate synthetase; Ade13p = adenylosuccinate lyase; Adk2p = mitochondrial GTP:AMP phosphotransferase; Adk1p = adenylate kinase; Imd2-4p = IMP dehydrogenase; Gua1p = GMP synthetase; Guk1p = guanylate kinase; PPAT = PRPP amidotransferase; TrifGART = trifunctional glycinamide ribonucleotide (GAR) transformylase; GARS = GAR synthase; GAR Tfase = GAR transformylase; AIRS = aminoimidazole ribonucleotide (AIR) synthase; FGAMS = formylglycinamidine ribonucleotide (FGAM) synthase; PAIC = phosphoribosylaminoimidazole carboxylase; CAIRS = carboxyaminoimidazole ribonucleotide (CAIR) synthase; SAICARS = succinylaminoimidazolecarboxamide ribonucleotide (SAICAR) synthase; ASL = adenylosuccinate lyase; ATIC = AICAR transformylase/IMP cyclohydrolase; AICAR Tfase = aminoimidazolecarboxamide ribonucleotide (AICAR) transformylase; IMPCH = IMP cyclohydrolase. (B) Assembly of PRPP synthetase subunits is enriched for Prs3p and Prs5p. GFP-tagged versions of the PRPP synthetase proteins (Prs1p, Prs2p, Prs3p, Prs4p, Prs5p) were grown in YPD to log phase, 1-, 3-, and 5-d time points and assayed for assembly formation. Representative images are shown below. (C) Only enzymes located at branch points (Ade4p and Ade16/17p) assemble into foci. GFP-tagged versions of purine biosynthetic enzymes acting in the middle of the pathway were grown under conditions identical to those indicated in B and assayed for assembly formation. Representative images are shown below. (D) Ade12p is the only enzyme forming foci in the ADP production branch. GFP-tagged versions of purine biosynthetic enzymes involved in ADP production were grown under conditions identical to those indicated in B and assayed for assembly formation. Representative images are shown below. (E) All subunits of the IMPDH complex assemble into foci. GFP-tagged versions of purine biosynthetic enzymes involved in GDP production were grown under conditions identical to those indicated in B and assayed for assembly formation. Representative images are shown below. Data are represented as means of at least three independent experiments; error bars indicate SEM. Images were taken from the culture condition with the highest degree of assembly.

FIGURE 4:

Only intracellular structures formed by metabolic enzymes performing the same reaction colocalize with each other. (A) Prs5p does not colocalize with any downstream enzyme in the de novo purine biosynthetic pathway. Dual fluorescent strains were grown to 5 d in YPD for imaging. (B) Only Ade16p and Ade17p foci show colocalization with each other. Growth conditions were identical to those indicated in A. (C) Imd4p fails to colocalize with its upstream enzyme (Ade4p, Ade16p) or its cross-pathway enzyme (Ade12p). Growth conditions were identical to those indicated in A. (D) Unlike in other eukaryotes, IMP dehydrogenase (Imd2-4p) does not colocalize with CTP synthetase (Ura7p). Growth conditions were identical to those indicated in A.

FIGURE 5:

Ade16p, Ade17p, and Imd3p are recruited into stress granules. (A) Ade16p, Ade17p, and Imd3p display high levels of colocalization with the stress granule marker Ded1p. Dual fluorescent strains were grown in YPD to 5 d, with the exception of Ade4p-GFP, which was examined at 1 d. (B) Enzymes in the purine biosynthetic pathway showed no colocalization with the processing body marker Edc3p. Growth conditions were identical to those indicated in A. (C) Ade16p colocalizes with the known stress granule–associated chaperone Hsp104p. Growth conditions were identical to those indicated in A. (D) Ade16p colocalizes with the known stress granule–associated chaperone Ssa1p. Growth conditions were identical to those indicated in A. (E) Quantification of colocalization of Ade16p with the chaperones Hsp104p and Ssa1p. Data are represented as means of at least three independent experiments; error bars indicate SEM.

FIGURE 6:

Coordinated structure formation of Prs5p and Ade4p controls pathway flux. (A) Prs3p, Prs5p, and Ade4p structures disassemble in response to the presence of fresh glucose. Cells expressing GFP-tagged purine biosynthetic enzymes were grown in YPD for 5 d, except for the ADE4::GFP strain (1 d), and then shifted into the indicated media, incubated for 30 min at 30°C, and visualized immediately. Protein levels were determined by Western blot analysis and were normalized to no- treatment samples (indicated below blots). (B) Prs5p and Ade4p have distinct triggers for structure formation. Yeast cells expressing GFP-tagged purine biosynthetic enzymes were grown to log phase in complete SD media, shifted into the indicated media for 30 min at 30°C, and counted immediately. (C) Deletion of downstream enzymes of Ade4p leads to increased structure formation of Ade4p. Wild-type and mutant cells expressing Ade4p-GFP were grown in YPD for 1 d at 30°C and scored for structure formation. Protein levels were determined by Western blot analysis and were normalized to the wild-type strain (indicated below blots). (D) Loss of feedback inhibition increases focus formation by Ade4p. Cells expressing wild-type Ade4p-GFP and Ade4p(K333Q)-GFP were grown to log phase in YPD and cells were scored for frequency of structure formation. Protein levels were determined by Western blot analysis and were normalized to the wild-type strain (indicated below blots). Data are represented as means of at least three independent experiments; error bars indicate SEM. (E) Model for the coordinating activity of Prs5p and Ade4p with regulated structure assembly statuses is illustrated.

FIGURE 7:

Filament formation of PRPP synthetase is evolutionarily conserved. Conservation of PRPP synthetase into filaments observed in Drosophila egg chambers (A), rat neurons (B), and human fibroblasts (C). The insert at the top left corner of each image is 3× magnified from the original image. PRPP synthase is stained with anti-PRPS1 (Covance; in red), and tubulin is detected with anti–tubulin-FITC (Sigma; in green).