Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells

Abstract

Metabolons, multiprotein complexes consisting of sequential enzymes of a metabolic pathway, are proposed to be biosynthetic “hotspots” within the cell. However, experimental demonstration of their presence and functions has remained challenging. We used metabolomics and in situ three-dimensional submicrometer chemical imaging of single cells by gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) to directly visualize de novo purine biosynthesis by a multienzyme complex, the purinosome. We found that purinosomes comprise nine enzymes that act synergistically, channeling the pathway intermediates to synthesize purine nucleotides, increasing the pathway flux, and influencing the adenosine monophosphate/guanosine monophosphate ratio. Our work also highlights the application of high-resolution GCIB-SIMS for multiplexed biomolecular analysis at the level of single cells.

Mechanical frustration of phase separation in the cell nucleus by chromatin

“…The thermodynamic ground state of two immiscible liquids is a single droplet of one liquid immersed in the other. Natural and synthetic condensates in cells, however, typically appear as dispersed droplets [9][10][11]. To test the stability of droplets in the cell, we used a novel optogenetic system [12,13] to create droplets in the nucleus ( Fig.…”

Metabolons, enzyme–enzyme assemblies that mediate substrate channeling, and their roles in plant metabolism

“…All of this evidence has been used to support the metabolic role of the purinosome, and although logical, it has the disadvantage of being circumstantial. In a very recent study, the Benkovic group ( Pareek et al., 2020 ) provided direct proof of this fact by demonstrating that the metabolite pathway (1) is functional and (2) promotes substrate channeling ( Figure 1 ). Although the best proof of the latter comes from isotope tracing experiments, quantitative analysis performed by ultra-high resolution mass spectral imaging provides strong support for the conclusions drawn from these analyses ( Pareek et al., 2020 ).…”

“…In a very recent study, the Benkovic group ( Pareek et al., 2020 ) provided direct proof of this fact by demonstrating that the metabolite pathway (1) is functional and (2) promotes substrate channeling ( Figure 1 ). Although the best proof of the latter comes from isotope tracing experiments, quantitative analysis performed by ultra-high resolution mass spectral imaging provides strong support for the conclusions drawn from these analyses ( Pareek et al., 2020 ). Currently, it is unknown whether a purinosome exists in plants; however, in plants, the pathway is mosaic in nature ( Sweetlove and Fernie, 2013 ), divided between the cytosol and plastid, so even if it exists, it is likely to be greatly different from that found in humans.…”

Chemical Analysis of Single Cells and Organelles

“…Pareek et al then used this GCIB to study the subcellular distribution of intact molecular ions of purine biosynthetic pathway intermediates and end nucleotides in HeLa cells. 140 …”