Septins function in exocytosis via physical interactions with the exocyst complex in fission yeast cytokinesis

Reviewer #1 (Public Review):

Summary:
In this manuscript, Singh, Wu and colleagues explore functional links between septins and the exocyst complex. The exocyst in a conserved octameric complex that mediates the tethering of secretory vesicles for exocytosis in eukaryotes. In fission yeast cells, the exocyst is necessary for cell division, where it localizes mostly at the rim of the division plane, but septins, which localize in a similar manner, are non-essential. The main findings of the work are that septins are required for the specific localization of the exocyst to the rim of the division plane, and the likely consequent localization of the glucanase Eng1 at this same location, where it is known to promote cell separation. In the absence of septins, the exocyst still localizes to the division plane but is not restricted to the rim. They also show some defects in the localization of secretory vesicles and glucan synthase cargo. They further propose that interactions between septins and exocysts are direct, as shown through Alphafold2 predictions (of unclear strength) and clean coIP experiments.

Strengths:
The septin, exocyst and Eng1 localization data are well supported, showing that the septin rim recruits the exocyst and (likely consequently) the Eng1 glucanase at this location. One major finding of the manuscript is that of a physical interaction between septins and exocyst subunits. Indeed, many of the coIPs supporting this discovery are very clear.

Weaknesses:
I am less convinced by the strength of the physical interaction of septins with the exocyst complex. Notably, one important open question is whether septins interact with the intact exocyst complex, as claimed in the text, or whether the interactions occur only with individual subunits. The two-hybrid and coIP data only show weak interactions with individual subunits, and some coIPs (for instance Sec3 and Exo70 with Spn1 and Spn4) are negative, suggesting that the exocyst complex does not remain intact in these experiments. Given the known structure of the full exocyst complex and septin filaments (at least in S. cerevisiae), the Alphafold2 predicted structure could be used to probe whether the proposed interaction sites are compatible with full complex formation.

The effect of spn1∆ on Eng1 localization is very clear, but the effect on secretory vesicles (Ypt3, Syb1) and glucan synthase Bgs1 is less convincing. The effect is small, and it is not clear how the cells are matched for the stage of cytokinesis.

Reviewer #2 (Public Review):

Summary:
This interesting study implicates the direct interaction between two multi-subunit complexes, known as the exocyst and septin complexes, in the function of both complexes during cytokinesis in fission yeast. While previous work from several labs had implicated roles for the exocyst and septin complexes in cytokinesis and cell separation, this study describes the importance of protein:protein interaction between these complexes in mediating the functions of these complexes in cytokinesis. Previous studies in neurons had suggested interactions between septins and exocyst complexes occur but the functional importance of such interactions was not known. Moreover, in baker's yeast where both of these complexes have been extensively studied - no evidence of such an interaction has been uncovered despite numerous studies which should have detected it. Therefore while exocyst:septin interactions appear to be conserved in several systems, it appears likely that budding yeast are the exception--having lost this conserved interaction.

Strengths:
The strengths of this work include the rigorous analysis of the interaction using multiple methods including Co-IP of tagged but endogenously expressed proteins, 2 hybrid interaction, and Alphafold Multimer. Careful quantitative analysis of the effects of loss of function in each complex and the effects on localization and dynamics of each complex was also a strength. Taken together this work convincingly describes that these two complexes do interact and that this interaction plays an important role in post Golgi vesicle targeting during cytokinesis.

Weaknesses:
The authors used Alphafold Multimer to predict (largely successfully) which subunits were most likely to be involved in direct interactions between the complexes. It would be very interesting to compare this to a parallel analysis on the budding yeast septin and exocyst complexes where it is quite clear that detectable interactions between the exocyst and septins (using the same methods) do not exist. Presumably the resulting pLDDT scores will be significantly lower. These are in silico experiments and should not be difficult to carry out.

Reviewer #3 (Public Review):

Septins in several systems are thought to guide the location of exocytosis, and they have been found to interact with the exocyst vesicle-tethering complex in some cells. However, it is not known whether such interactions are direct or indirect. Moreover, septin-exocyst physical associations were not detected in several other systems, including yeasts, making it unclear whether such interactions reflect a conserved septin-exocytosis link or whether they may missed if they depend on septin polymerization or association into higher-order structures. Singh et. al., set out to define whether and how septins influence the exocyst during S. pombe cytokinesis. Based on three lines of evidence, the authors conclude that septins directly bind to exocyst subunits to regulate localization of the exocyst and vesicle secretion during cytokinesis.
The conclusions are consistent with the data presented, but some interpretations need to be clarified and extended:

(1) The first line of evidence examines septin and exocyst localization during cytokinesis in wild-type and septin-mutant or exocyst-mutant yeast. Quantitative imaging convincingly shows that the detailed localization of the exocyst at the division site is perturbed in septin mutants, and that this is accompanied by modest accumulation of vesicles and vesicle cargos. Whether that is sufficient to explain the increased thickness of the division septum in septin mutants remains unclear.

(2) The second line of evidence involves a comprehensive Alphafold2 analysis of potential pair-wise interactions between septin and exocyst subunits. This identifies several putative interactions in silico, but it is unclear whether the identified interaction surfaces would be available in the full septin or exocyst complexes.

(3) The third line of evidence uses co-immunoprecipitation and yeast two hybrid assays to show that several physical interactions predicted by Alphafold2 can be detected, leading the authors to conclude that they have identified direct interactions. However, both methods leave open the possibility that the interactions are indirect and mediated by other proteins in the fission yeast extract (co-IP) or budding yeast cell (two-hybrid).

(4) Based on prior studies it would be expected that the large majority of both septins and exocyst subunits are present in cells and extracts as stoichiometric complexes. Thus, one would expect any septin-exocyst interaction to yield associations detectable with multiple subunits, yet co-IPs were not detectded in some combinations. It is therefore unclear whether the interactions reflect associations between fully-formed functional complexes or perhaps between transient folding intermediates.