Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 190 (4), 541-51

Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster

Affiliations

Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster

Chalongrat Noree et al. J Cell Biol.

Abstract

The discovery of large supramolecular complexes such as the purinosome suggests that subcellular organization is central to enzyme regulation. A screen of the yeast GFP strain collection to identify proteins that assemble into visible structures identified four novel filament systems comprised of glutamate synthase, guanosine diphosphate-mannose pyrophosphorylase, cytidine triphosphate (CTP) synthase, or subunits of the eIF2/2B translation factor complex. Recruitment of CTP synthase to filaments and foci can be modulated by mutations and regulatory ligands that alter enzyme activity, arguing that the assembly of these structures is related to control of CTP synthase activity. CTP synthase filaments are evolutionarily conserved and are restricted to axons in neurons. This spatial regulation suggests that these filaments have additional functions separate from the regulation of enzyme activity. The identification of four novel filaments greatly expands the number of known intracellular filament networks and has broad implications for our understanding of how cells organize biochemical activities in the cytoplasm.

Figures

Figure 1.
Figure 1.
Identification of nine proteins capable of filament formation in S. cerevisiae. (A) Nine filament-forming proteins were identified by visual screening of the S. cerevisiae GFP strain collection: Glt1p (glutamate synthase), Psa1p (GDP-mannose pyrophosphorylase), Ura7p (CTP synthase), Ura8p (CTP synthase), Gcd2p (eIF2B-δ), Gcd6p (eIF2B-ε), Gcd7p (eIF2B-β), Gcn3p (eIF2B-α), and Sui2p (eIF2-α). (B) The nine proteins that are capable of forming filaments were found to reside in four distinct filaments. All images are of cells grown to saturation except for subunits of the eIF2/2B complex, which were from log-phase cultures. These conditions were chosen because they maximized filament formation for the respective subunits.
Figure 2.
Figure 2.
Regulation of filament formation. (A) Filament formation is not dependent on either HSP104 or RNQ1. (B) Overexpression of Hsp104p does not affect the formation of Glt1p, Psa1p, or Ura7p filaments. (C) Media lacking glucose strongly induces Ura7p filaments. (D) The addition of media containing glucose triggers disassembly of Ura7p filaments. (E) Treatment with sodium azide causes an increase in Psa1p and Ura7p filaments. (F) Treatment with the translation inhibitor cycloheximide decreases the number of Gcd2p filaments. (G) Exposure of cells to 4°C has no effect on filament formation. (H) Treatment with the kinase inhbitor staurosporine increases Psa1p and Ura7p filaments. (A–H) Error bars represent standard error of the mean. Dashed lines mark the position on the graph where there is no change relative to the reference condition.
Figure 3.
Figure 3.
Filament formation is evolutionarily conserved. (A) A single confocal section of a Drosophila egg chamber. GFP–CTP synthase (green) is present in small filaments (yellow arrowhead) along the plasma membrane and in large filaments in both the somatic follicle cells (red arrowhead) and nurse cells (white arrowhead). Actin is red, and DNA is blue. (B) A projection of multiple confocal sections of an egg chamber stained with anti–CTP synthase antibody. Large filaments are present in the germline (white arrow) as well as in the somatic follicle cells (red arrow). Actin is red, and CTP synthase is green. (C) In the adult Drosophila gut, GFP–CTP synthase (green) labels filaments (arrowheads) in cells clustered near the presumptive gut stem cell, labeled with Delta (red). DNA is blue.
Figure 4.
Figure 4.
CTP synthase self-assembles in axons but not in dendrites. CTP synthase (CTPS) filaments are present in axons (arrowheads) but not dendrites. CTP synthase does not form filaments or foci in dendritic processes. (A–C) MAP2c (A), CTP synthase (B), and a merge (C) are shown. MAP2c is red, and CTP synthase is green. CTP synthase forms filaments and foci in axons (arrowheads). (D–F) Tau (D), CTP synthase (E), and a merge (F) are shown. Tau is red, and CTP synthase is green.
Figure 5.
Figure 5.
End product inhibition promotes CTP synthase filament formation. (A) The E161K mutation causes a 20-fold decrease in Ura7p filament formation. (B) Treatment with CTP and ATP increases Ura7p self-assembly into foci. (A and B) Error bars represent standard error of the mean. Dashed lines mark the position on the graph where there is no change relative to the reference condition.

Similar articles

See all similar articles

Cited by 108 PubMed Central articles

See all "Cited by" articles

References

    1. An S., Kumar R., Sheets E.D., Benkovic S.J. 2008. Reversible compartmentalization of de novo purine biosynthetic complexes in living cells. Science. 320:103–106 10.1126/science.1152241 - DOI - PubMed
    1. Aronow B., Ullman B. 1987. In situ regulation of mammalian CTP synthetase by allosteric inhibition. J. Biol. Chem. 262:5106–5112 - PubMed
    1. Baudin A., Ozier-Kalogeropoulos O., Denouel A., Lacroute F., Cullin C. 1993. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 21:3329–3330 10.1093/nar/21.14.3329 - DOI - PMC - PubMed
    1. Buszczak M., Paterno S., Lighthouse D., Bachman J., Planck J., Owen S., Skora A.D., Nystul T.G., Ohlstein B., Allen A., et al. 2007. The carnegie protein trap library: a versatile tool for Drosophila developmental studies. Genetics. 175:1505–1531 10.1534/genetics.106.065961 - DOI - PMC - PubMed
    1. Campbell S.G., Hoyle N.P., Ashe M.P. 2005. Dynamic cycling of eIF2 through a large eIF2B-containing cytoplasmic body: implications for translation control. J. Cell Biol. 170:925–934 10.1083/jcb.200503162 - DOI - PMC - PubMed

Publication types

MeSH terms

Feedback