The Majority of the Saccharomyces cerevisiae Septin Complexes Do Not Exchange Guanine Nucleotides

Abstract

We show here that affinity-purified Saccharomyces cerevisiae septin complexes contain stoichiometric amounts of guanine nucleotides, specifically GTP and GDP. Using a 15 N-dilution assay read-out by liquid chromatography-tandem mass spectrometry, we determined that the majority of the bound guanine nucleotides do not turn over in vivo during one cell cycle period. In vitro, the isolated S. cerevisiae septin complexes have similar GTP binding and hydrolytic properties to the Drosophila septin complexes (Field, C. M., al-Awar, O., Rosenblatt, J., Wong, M. L., Alberts, B., and Mitchison, T. J. (1996) J. Cell Biol. 133, 605-616). In particular, the GTP turnover of septins is very slow when compared with the GTP turnover for Ras-like GTPases. We conclude that bound GTP and GDP play a structural, rather then regulatory, role for the majority of septins in proliferating cells as GTP does for ␣-tubulin.

Some assembly required: yeast septins provide the instruction manual

“…In addition, each septin (Cdc3, Cdc10, Cdc11 and Cdc12) can self-associate. In this regard, the apparent molecular weight of recombinant complexes isolated at a moderate salt concentration (0.25 M) is consistent with two copies of each septin in a 1:1:1:1 stoichiometry [10], whereas the amount of Cdc11 in native complexes that are isolated at higher salt concentrations (0.5-1.0 M) is substoichiometric [8,38]. Likewise, in high-salt conditions, the amount of Shs1 recovered in native complexes is substoichiometric [39].…”

“…However, the rates of nucleotide exchange and hydrolysis observed in vitro are slow. This intrinsic property seems unaffected by cellular factors because the turnover of GTP in septin complexes in vivo, judged by mass spectrometry, is also very slow, at least in budding yeast [38]. Using radioactively labeled nucleotides (or fluorescent or photo-activatable analogs), it has been shown that some septin subunits, for example yeast Cdc10 and Cdc12, and fly Pnut and Sep1, bind GTP whereas others, for example yeast Cdc3 and Cdc11, and fly Sep2, do not [7,28].…”

“…The GDP:GTP ratio in septin complexes (~2) seems to be conserved [7,9,38]. Given the number of different septins in these hetero-oligomers, the effects of nucleotide are potentially even more complicated than in polymerization of αβ tubulin heterodimers into microtubules [60].…”

“…Two observations do not support such a regulatory role for formation and breakdown of the septin filaments at the bud neck in S. cerevisiae, at least. First, using isotopic labeling of cells and analysis of the nucleotide content of isolated septin hetero-oligomers by liquid chromatography and tandem mass spectrometry, it appears that the bound guanine nucleotides show either little or no turnover during one cell-cycle period [38]. Thus, GTP binding and hydrolysis within septin hetero-oligomers is too slow to account for the rate of assembly and disassembly of the filaments that is observed in vivo at the collar.…”

Kinetic partitioning during de novo septin filament assembly creates a critical G1 “window of opportunity” for mutant septin function

“…Yeast septins are exceedingly long-lived proteins that are recycled through mitotic cell divisions. 20 In early G1, mother and bud disassemble their "old" septin rings and thereby generate a supply of pre-assembled octamers that are competent to incorporate into new higher-order structures during late G1/early S. 20,21 However, successful construction of an intact ring of filaments at the beginning of S phase presumably also requires synthesis and assembly of additional, new octamers. We thus suspected that G1 represents a critical window of time for new septin hetero-oligomer assembly, wherein a WT and a slowly-folding mutant allele compete for incorporation into hetero-oligomers, and mutant molecules must achieve oligomerization-competent conformations in order to support septin function in cells with no other allele available.…”