A draft of the human septin interactome

Abstract

Background: Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins.

Methodology/principal findings: Here, we performed yeast two-hybrid screens with human septins 1-10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments.

Conclusions/significance: If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the "group rule", i.e. members of the same group (e.g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.

Figure 1. Summary of protein-protein interactions found for human septins 1-10.

The values are given as a percentage of clones from the total number of confirmed interacting clones sequenced and identified. See color code for septin specification. Septins are grouped according to the four groups SEPT2, SEPT3, SEPT6 and SEPT7 from left to right.

Figure 2. Three dimensional column diagram of the number of clones of septin prey proteins fished by septin bait proteins in group wise organization.

Number of fished clones for each prey septin (Y-axis), was plotted against bait (X-axis) and prey septins (Z-axis), where the septins have been ordered in a group wise fashion (See Table S1 for raw data of clone numbers). The whole distribution is significantly (p = 0.0004998) different from a random distribution, as can also be verified visually, since the clone numbers group into “islands” in between various blank areas. When we compared groups in a one-to-one and reciprocal fashion we obtained significant p-values (p<0.001), suggesting a non-random distribution, for the following pairs of groups: group 2 vs. group 6, group 6 vs. group 3/9, group 6 vs. group 7, and group 3/9 vs. group 7. Furthermore, we can still analyze the occurrence of interactions among septins of the same group. The result in this case is obvious: members of groups 6, 3/9 and 7 never interacted with themselves or with other members of the same group. The only exception is group 2, which members tended to interact with other group 2 members. See also Fig. 3.

Figure 3. Representation of the two-hybrid septin-septin results in the light of the format of the trimer/hexamer SEPT7/SEPT6/SEPT2 – SEPT2/SEPT6/SEPT7 .

Assuming that members of the same group may serve as substitutes and taking into account the structural arrangement found for the crystal of the trimer/hexamer SEPT 7/6/2, interesting observations can be made. The bait septins employed in the two-hybrid system are given on the left. In green in the schematic figure the preferentially found prey septins are indicated and assigned to likely positions in the hexamer scheme. The three dimensional column diagrams on the right refer to Fig. 2. The data that differ significantly from a random distribution have been circled to indicate the experimental basis on which each structural arrangement (monomer interfaces) is based. From top to bottom: (a) is based on the following statistical comparison: group 6 vs. group 2 (no random distribution: p< 0.001), group 6 vs. group 3/9 (p<0.001), group 6 vs. 7 (p<0.001). (b): group 2 vs. group 6 (p<0.001), (c): group 3/9 vs. group 6 (p<0.001), group 7 vs. group 9/3 (p<0.001), (d): group 7 vs. group 6 (p<0.001), group 7 vs. group 9/3 (p<0.001). None of the septins fished members of its own family, except group two members (b). The group pairings with statistically significant clone distributions were indicated at the right side of the figure (e.g. 6×7, 3/9×6 etc.). By comparison of the letters color codes it can be seen that all of these group pairs were reciprocal. For example bait septin 6 group fished group 2 septins (a) and vice versa (b) (p <0.001). As initially proposed by Kinoshita there may be substitutions among different members of the same septin group. The sequence of listed septins from left to right reflects a descending order of frequency of clones with septins that were found to interact (e.g. for SEPT6 bait: 9,3,7 and 5,1,4,2).

Figure 4. Schematic representation of possible septin-septin interactions within putative filaments, based on a combination of our yeast two hybrid assay's data and previously published data.

(A) the canonical or standard filament taken from the crystal structure of the 7-6-2 complex showing the trimeric and hexameric cores and the NC and G interface. The 7-6 dimer is believed to be stabilized by a long coiled coil at an NC interface, whilst the 2-2 dimer is similarly stabilized by a short coiled coil. (B) possible arrangement for a filament composed of members from the SEPT3, SEPT6 and SEPT2 groups. SEPT3 group contains both septin 3 and 9. This arrangement would leave the SEPT6 coiled coil unpaired and “free” for interaction with other coiled-coil containing proteins (see text for details). (C) possible arrangement for a filament composed of members from the SEPT3, SEPT6 and SEPT7 groups. (D) possible position for SEPT9 compatible with the observed yeast two-hybrid data and data from the literature (see also Fig. 3c,d).

Figure 5. Non-septin interactors of the septins 1-10 grouped by functional categories.

The group wise clustering of functional contexts is evident. The most predominant functions are emphasized only in order not to pollute the figure with excessive information. Septins are shown clustered into the four groups: SEPT2, SEPT3, SEPT6 and SEPT7 from top to bottom in different colors.