Eukaryotic GPN-loop GTPases paralogs use a dimeric assembly reminiscent of archeal GPN

Abstract

GTPases are molecular switches that regulate a wide-range of cellular processes. The GPN-loop GTPase (GPN) is a sub-family of P-loop NTPase that evolved from a single gene copy in archaea to triplicate paralog genes in eukaryotes, each having a non-redundant essential function in cell. In Saccharomyces cerevisiae, yGPN1 and yGPN2 are involved in sister chromatid cohesion mechanism, whereas nothing is known regarding yGPN3 function. Previous high-throughput experiments suggested that GPN paralogs interaction may occur. In this work, GPN|GPN contact was analyzed in details using TAP-Tag approach, yeast two-hybrid assay, in silico energy computation and site-directed mutagenesis of a conserved Glu residue located at the center of the interaction interface. It is demonstrated that this residue is essential for cell viability. A chromatid cohesion assay revealed that, like yGPN1 and yGPN2, yGPN3 also plays a role in sister chromatid cohesion. These results suggest that all three GPN proteins act at the molecular level in sister chromatid cohesion mechanism as a GPN|GPN complex reminiscent of the homodimeric structure of PAB0955, an archaeal member of GPN-loop GTPase.

The Gpn3 Q279* cancer‐associated mutant inhibits Gpn1 nuclear export and is deficient in RNA polymerase II nuclear targeting

“…Importantly, the functional changes observed in Gpn3 Q279* mostly disappeared after disrupting the newly formed PDZ-binding motif, even by eliminating one more amino acid from Gpn3 Q279*, supporting our proposal that it is the acquisition of this motif, and not the loss of the C-terminal integrity what causes the observed functional changes in Gpn3 Q279*. Given that chromosome instability is a defining feature of cancer cells, it is worth noting that, at least in the yeast S. cerevisiae, Gpn3 plays an important role in chromosome segregation [6,20]. If this function is conserved in human cells, as is Gpn3 involvement in RNAPII nuclear targeting [4,6], then it would be expected that, regardless of the moment when one copy of GPN3 disappears and the Gpn3 Q279* mutant is generated during the development of the tumor, the appearance of this Gpn3 mutant might be a physiologically relevant event, as the reduction in gene dosage and the functional alterations caused by the Q279* mutation may decrease Gpn3 function, which in turn would speed the chromosome instability characteristic of cancer cells.…”

Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

“…In the yeast Saccharomyces cerevisiae, deletion of the GPN1 homolog Npa3 or its paralogs GPN2 (YOR262W) and GPN3 (YLR243W) is lethal (9), indicating essential, nonredundant functions of these enzymes. Homo-and heterodimerization of GPN1 and its paralogs were reported previously (10)(11)(12). Npa3 contains a nuclear export sequence (NES) (residues 286 to 295) (13), consistent with the predominant cytoplasmic localization of Npa3 in yeast (14,15) and GPN1 in human cells (7,13,16,17).…”

Gpn1 and Gpn3 associate tightly and their protein levels are mutually dependent in mammalian cells

“…This observation has led to the proposal that Gpn proteins belong to a group of GTPases activated by dimerization [19]. Given the similarity in amino acid sequence between the GTPase domain of Gpn1 and Gpn3 [17], is possible that in eukaryotic cells, where all or different combinations of all three GTPases Gpn (Gpn1, Gpn2 and Gpn3) may be expressed, they could actually associate and function as heterodimers.…”