1

Sho1p connects the plasma membrane with proteins of the

cytokinesis network via multiple isomeric interaction states

Karolina Labedzka, Chen Tian, Ute Nussbaumer, Steffi Timmermann, Paul Walther+,

Judith Müller, and Nils Johnsson#

Institute of Molecular Genetics and Cell Biology, Department of Biology, Ulm

University, James-Franck-Ring N27, D-89081 Ulm, Germany + Central Facility for Electron Microscopy, Ulm University

Contact: Nils Johnsson, Institute of Molecular Genetics and Cell Biology, Department of

Biology, Ulm University, James-Franck-Ring N27, D-89081 Ulm, Germany. Phone: + 49

731 50 36300; Fax : + 49 731 50 36302; e-mail: nils.johnsson@uni-ulm.de

# Corresponding author

Running title: Molecular description of the HICS-complex

Key words: Constraint interaction networks /cytokinesis/protein interaction/split-

ubiquitin/systems biology/

© 2012. Published by The Company of Biologists Ltd.Jo

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JCS online publication date 23 May 2012

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Summary

A molecular understanding of cytokinesis requires the detailed description of

the protein complexes that perform central activities during this process. The

proteins Hof1p, Cyk3p, Inn1p, and Myo1p each represent one of the four

genetically defined and partially complementing pathways of cytokinesis in the

yeast Saccharomyces cerevisiae. Here we show that the osmosensor Sho1p is

required for correct cell-cell separation. Shortly before cytokinesis Sho1p

sequentially assembles with Hof1p, Inn1p, and Cyk3p, into a complex (HICS-

complex) that might help to connect the membrane with the actin-myosin ring.

The HICS-complex is formed exclusively via the interactions between three SH3

domains located in Cyk3p, Hof1p, and Sho1p, and five acceptor sites found in

Cyk3p, Hof1p, and Inn1p. Due to the overlapping binding specificities of its

members the HICS-complex is best described as ensembles of isomeric

interaction states that precisely coordinate the different functions of the

interactors during cytokinesis.

Introduction

To separate mother from daughter, yeast cells undergo a well-orchestrated set of

biochemical reactions: contraction of the actin-myosin ring (AMR), ingression and

fusion of the plasma membrane, synthesis of a chitin-containing primary and a

secondary septum (PS, SS), and, finally, the partial degradation of the primary

septum to liberate both cells from each other (Balasubramanian et al., 2004; Bi, 2001;

Schmidt et al., 2002).

Although the enzymes responsible for performing these activities are well known it

remains an unresolved issue how these enzymes are regulated, and how their

individual contributions are integrated in time and space to form a precisely operating

cell-cell separation machinery (Schmidt et al., 2002; Tolliday et al., 2001). One

outstanding question is how the AMR is connected to the membrane to transform its

contraction into membrane ingression. Candidate proteins for this linkage are the

proteins Hof1p and Inn1p. Both are known to be involved in the correct progression

of cell separation (Kamei et al., 1998; Lippincott and Li, 1998; Sanchez-Diaz et al.,

2008; Vallen et al., 2000). Their specific roles have, however, not yet been precisely

defined. Hof1p may directly connect the membrane with the AMR during contraction

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as the phenotypes of its deletion as well as its localization during cytokinesis are

compatible with such a role (Meitinger et al., 2011). In addition, Hof1p contains at its

N-terminus a F-BAR domain. Other members of the family of F-BAR-containing

proteins were shown to bind to the actin cytoskeleton and to simultaneously bind and

bend membranes (Itoh et al., 2005; Rao et al., 2010). However, both features have not

been demonstrated for Hof1p. Hof1p was shown to contact via its C-terminal SH3

domain Inn1p, and Cyk3p, a further SH3 domain-containing protein (Jendretzki et al.,

2009; Korinek et al., 2000; Nishihama et al., 2009). Inn1p is essential for cytokinesis.

Its N-terminal C2-domain was postulated to bind to membranes and the co-

precipitation with Iqg1p, a member of the AMR, was taken as evidence for an

important role in linking the membrane to the AMR (Sanchez-Diaz et al., 2008). An

alternative model suggesting that Inn1p and Cyk3p activate and coordinate the

formation of the SS and PS recently questioned this conclusion (Nishihama et al.,

2009).

One goal of protein-protein interaction analyses is a detailed interaction map that

helps to navigate through the simultaneously or sequentially occurring events during

cellular processes such as cytokinesis (Vidal et al., 2011). In yeast, many of the

relevant protein interactions have probably already been detected by genome-wide

screens and are easily accessible in public databases. For example, novel interaction

partners of Hof1p were recently identified through a systematic mapping of the

interaction partners of all yeast genome-encoded SH3 domains by phage display,

peptide spotting, and two hybrid analysis (Tonikian et al., 2009). However, a direct

incorporation of large-scale protein interaction data into models of cytokinesis is not

straightforward, as the data sets are still incomplete and potentially erroneous.

Furthermore, most of the applied high throughput methods are performed under

conditions rather distant from the situation within the living cell and thus lack one

level of information required for realistic and predictive model building.

We recently introduced a Split-Ubiquitin-based approach (Split-Ub) of collecting and

structuring protein-protein interactions to build constraint protein interaction

networks (CIN) (Hruby et al., 2011; Johnsson and Varshavsky, 1994; Müller and

Johnsson, 2008). By experimentally determined constraints these networks allow to

discriminate between different interaction states. Interaction states are defined as the

subset of all detected interactions of a local network that are simultaneously allowed.

This feature of CINs reduces the number of possible combinations of protein

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interactions within a network and thus provides better frameworks for

mechanistically meaningful models describing their functions and workings (Hruby

et al., 2011).

Here we report on a local protein interaction network around the integral membrane

protein and osmosensor Sho1p. Sho1p, together with other membrane proteins,

senses conditions of high osmolarity by a still unknown mechanism. It then serves as

a platform for the assembly of the MAP kinase pathway that transforms the signal

into a cellular response (Hohmann, 2009; Tatebayashi et al., 2007). Our derived CIN

suggests an additional unexpected role of Sho1p in cytokinesis by linking via its SH3

domain the known members of the cytokinesis machinery Hof1p, Inn1p, and Cyk3p

to the plasma membrane.

Results

Novel binding partners of Sho1p

A Split-Ub-based systematic interaction screen against an array of 383 Nub-fusions

revealed novel and known interaction partners of the Cub-RUra3 fusion of the

osmolarity sensor Sho1p (Sho1CRU; Figure 1A, Table S1). Although this Nub-array

is enriched in fusion genes involved in polarized growth, cytokinesis, and stress

responses we noted among the binding partners a surprising predominance of proteins

involved in cytokinesis (Hruby et al., 2011). To exclude in a first step those of the

detected contacts that merely reflect the co-localization within the membrane rather

than a specific protein-protein interaction, we repeated the experiment with a

truncated version of Sho1p. This mutant still harbored its four trans-membrane

spanning elements but lacked the C-terminal SH3 domain (Sho11-297). Comparison of

the obtained interaction profiles for Sho1CRU and Sho1p1-297CRU left as SH3-

dependent binding partners the MAP kinase kinase of the osmolarity pathway Pbs2p,

the cytokinesis proteins Hof1p and its N-terminally truncated version Hof198-669,

Inn1p, Cyk3p and its N-terminally truncated allele Cyk369-885, the scaffold protein

Msb1p, the chitin synthase Chs3p, Sho1p, and the stimulator of exocytosis Sro7p

(Table S1 and Figure 1A) (Hao et al., 2007; Tonikian et al., 2009). To prove a direct

involvement of the SH3 domain of Sho1p (SH3Sho1, residues 298-367) in the

potentially cytokinesis-relevant interactions, we precipitated from yeast extracts

MYC-tagged versions of Cyk3p, Inn1p, and Hof1p with the bacterially expressed and

immobilized SH3Sho1. As shown in Figure 1B all proteins with a well-established role

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in cytokinesis bound the SH3 domain of Sho1p. These interactions were abolished

when the SH3 domain was deleted from the full-length Sho1p (Figure 1A).

Dissecting the SH3Sho1 binding sites

Besides the full-length Cyk3p the Nub-array contains two N-terminally truncated

versions missing the N-terminal SH3 domain (Nub-Cyk369-885), or the N-terminal 196

residues (Nub-Cyk3196-885), respectively. Remarkably, Sho1CRU still interacted with

Nub-Cyk369-885 but not with Nub-Cyk3196-885, although both Nub-fragments of Cyk3p

were expressed at similar levels in the yeast (Figures 1A, S1). This finding locates the

binding site to SH3Sho1 between residues 69 and 196 of Cyk3p. This sequence harbors

a proline-rich stretch between residues 183 and 191 that resembles the binding motif

of Pbs2p for Sho1p (Maeda et al., 1995). We expressed residues 160 to 210 of Cyk3p

as a fusion to O6-Alkylguanine-DNA alkyltransferase in E.coli (AGT; AGT-Cyk3160-

210). AGT-Cyk3160-210 directly interacted with the purified GST-fusion of SH3Sho1

(Figure 1C). Replacement of the prolines in P185PLPPLP191 of Cyk3p (AGT-Cyk3160-

210ΔP) completely abolished the binding to GST-SH3Sho1 (Figure 1C).

Two findings restricted the binding site of SH3Sho1 to the region between residues 98

and 592 of Hof1p: The Nub-fusion of Hof198-669 lacking its FCH domain still bound to

Sho1CRU, and bacterially expressed SH3Sho1 precipitated Hof1p lacking the C-

terminal SH3 domain (Nub-Hof11-592)(Figure S1). This central region of Hof1p

harbors at positions 333-338 and 386-392 two proline-rich stretches (P1, P2) as

prospective docking sites for SH3Sho1 (Figure 1D). Pull-down assays with bacterially

expressed Hof1p fragments showed a specific and direct interaction between the

purified SH3Sho1 and Hof1269-349, harboring P1. Hof1366-423, containing P2, did not

interact with SH3Sho1 (Figure 1D).

Inn1p is a further binding partner of Sho1p with a well-established role in cytokinesis

(Figures 1A, B) (Nishihama et al., 2009; Sanchez-Diaz et al., 2008; Tonikian et al.,

2009). Inn1p contains multiple proline-rich motifs that are known to bind to different

SH3 domain-containing proteins including Hof1p and Cyk3p (Figure 3C) (Nishihama

et al., 2009; Schreiter et al. 2009; Tonikian et al., 2009; Xin et al. 2009). To identify

the motif in Inn1p responsible for binding to Sho1p, we expressed in E. coli

fragments of Inn1p as AGT-fusion proteins, each encompassing different proline-rich

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stretches. Precipitations with purified GST-SH3Sho1 identified the most C-terminally

located proline-rich motif of Inn1p as the ligand for SH3Sho1 (Figure 1E).

Msb1p was originally identified as a suppressor of certain bem1 and cdc24 alleles and

was later shown to stimulate the synthesis of β1-3 Glucan in the extracellular matrix

(Bender and Pringle, 1989; Sekiya-Kawasaki et al., 2002). Inspection of the Msb1p

sequence revealed at position 6-12 a region with a close resemblance to the proline-

rich motif in Pbs2p (Maeda et al., 1995). We precipitated the N-terminal 39 residues

of Msb1p as bacterially expressed AGT-fusion (Msb11-39-AGT) with the purified

GST-SH3Sho1. The efficient binding supports our hypothesis that this motif indeed

represents the in vivo binding site for Sho1p (Figure 1 F).

In summary, this detailed protein interaction analysis of Sho1p identifies as binding

sites of its SH3-domain four new proline-rich motifs in four different proteins. Three

of these proteins, namely Hof1p, Cyk3p, and Inn1p, are known to play an important

role in cytokinesis (Figure 1G).

The architecture of the HICS-complex: Identification of new binding partners

and dissection of the binding sites

Our interaction analysis suggested that Hof1p, Cyk3p, Inn1p, and Sho1p might act

together in a complex. To identify additional binding partners of this putative

complex we screened Cyk3CRU, Hof1CRU, and Inn1CRU against our Nub-array

(Table S1). We could confirm the pairwise interactions between all three proteins. In

addition, Hof1p and Cyk3p but not Inn1p were found to interact with Nba1p. Cdc3p

was a unique interaction partner of Hof1p. Slt2p, Tpk1p, and Bud14p bound

exclusively to Cyk3p. Sla1p, Ubc9p as well as Cdc14p interacted only with Inn1p

(see Table S1, Figures 2A, 3A, B).

To delineate the individual binding sites for their ligands, we created different

fragments of Cyk3p, Hof1p, and Inn1p as CRU fusions. The N-terminal fragment of

Cyk3 containing both the SH3 domain as well as the SH3Sho1 binding site (Cyk31-

267CRU) still interacted with Nub-Hof1p, -Inn1p, and -Nba1p but not any longer with

Nub-Slt2p and -Bud14p (Figure 2A). The isolated SH3Cyk3 (Cyk31-79CRU) retained

the ability to interact with Nub-Inn1p and -Nba1p, whereas a Cyk3 fragment

harboring the isolated proline-rich stretch (Cyk3119-267CRU) only interacted with Nub-

Hof1p and its N-terminally truncated variant Hof198-669 (Figure 2A).

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To further prove that the proline-rich stretch of Cyk3p is indeed the binding site for

SH3Hof1, we mutated this site in the N-terminal Cyk3p fragment (Cyk31-267ΔPCRU)

and tested it against different Nub-fragments of Hof1p (Figure 2C). This experiment

unambiguously mapped the interaction between Hof1p and Cyk3p to SH3Hof1 and the

proline-rich stretch between residues 183 and 191 of Cyk3p (P1). This interaction

was direct as the purified GST-Hof1342-669 precipitated from E.coli extracts AGT-

Cyk3160-210 and to a much lower degree AGT-Cyk3160-210ΔP (Figure 2D). The small

amount of precipitated AGT-Cyk3160-210ΔP might have been caused by the high

concentrations of both proteins in the assay. In these in vitro experiments we had to

N-terminally extend the SH3 domain of Hof1p (GST-Hof1342-669 (GST-SH3Hof1)) as a

smaller fragment including SH3Hof1 (residues 490-669) showed no in vitro activity

towards any of its ligands (our unpublished observation).

Do Slt2p and Bud14p bind to an autonomous domain within the C-terminal region of

Cyk3p? We assayed a CYK3 fragment missing SH3Cyk3 (Cyk3119-885CRU) and the

same fragment harboring a mutated P1 (Cyk3119-885ΔPCRU) (Figure 2B). Nub-Bud14p

and -Slt2p still interacted with both Cyk3CRU fusions, whereas Nub-Hof1p was only

bound by Cyk3119-885CRU. Additionally, we detected a weak affinity of both

Cyk3CRU fragments to the N-terminally truncated version Nub-Cyk3196-885 (Figure

2B). As homodimerization of full-length Cyk3p was never observed we did not

include this interaction in the summary of the obtained results (Figure 2E).

Similar to Sho1CRU, Hof1CRU still interacted with Nub-Cyk369-885 but not with Nub-

Cyk3196-885 (Figure 3A). The experiment provided additional evidence that the same

proline-rich stretch in Cyk3p served as binding site for Hof1p and Sho1p. A C-

terminal fragment of Hof1p (Hof1343-669CRU) harboring the SH3 but not its F-BAR

domain still interacted with Nub-Cyk3p, -Cyk369-885, -Inn1p, and Nub-Nba1p (Figure

3A).

Inn1p displays four proline-rich stretches within its sequence (Figure 3C) (Nishihama

et al., 2009). The interactions of Inn1p with Cyk3p and Hof1p were already mapped

to the proline-rich motifs P2 and P4 of Inn1p, respectively (Nishihama et al., 2009).

To independently map the binding sites of some of the herein found interaction

partners within the Inn1p sequence we tested different Inn1CRU-fragments against a

selection of Nub-fusions (Figure 3B). The measured interaction between Nub-Cyk3p

and Inn11-211 confirmed previous studies (Figure 3B) (Nishihama et al., 2009). Hof1p

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and Hof198-669 interacted with P4 (Inn1367-409) and P3 (Inn11-373). Sla1p and Cdc14p

interacted exclusively with Inn1367-409 (Figure 3B). This dissection predicted that the

SH3 domains of Hof1p, Sla1p, and Sho1p might compete for binding the C-terminal

P4 motif of Inn1p (Figure 3C). The absence of a SH3 domain lets us assume that the

binding of Cdc14p is not mediated by P4 and thus not directly influenced by the

presence of the other ligands (Figure 3C). A cluster of in vivo phosphorylation sites in

Inn1367-409 might constitute the docking site for Cdc14p (Bodenmiller et al., 2008).

The identification of non-overlapping binding sites supports the existence of a so far

unknown protein complex consisting of the four core subunits Hof1p, Inn1p, Cyk3p

and Sho1p (HICS-complex, Figure 3C).

Membrane attachment of SH3Sho1 is a prerequisite for ligand binding in vivo

The in vitro precipitations of the proline-rich motifs with the isolated SH3Sho1 were

performed under high concentrations of both reactants (Figure 1 C-F). In vivo, Hof1p,

Cyk3p, and Inn1p locate to the bud neck below the membrane during cytokinesis

while Pbs2p is distributed in the cytosol (Jendretzki et al., 2009; Nishihama et al.,

2009; Sanchez-Diaz et al., 2008). Are the interactions between SH3Sho1 and its

ligands restricted to the bud neck? We compared the in vivo interaction profile of the

CRU-fusion of SH3Sho1 (SH3Sho1CRU) with that of the full length Sho1CRU to

elucidate the influence of the cellular localization on its interactions (Figure 4A).

Strikingly, only the Nub-fusion of Pbs2p was found as an interaction partner of the

cytosolic SH3Sho1CRU in this in vivo assay, implicating the membrane association of

SH3Sho1 as a prerequisite for its effective interaction with Inn1p, Cyk3p, Msb1p, and

Hof1p (Figure 4A). Similar in vivo measurements with the SH3 domains of Cyk3p

and Hof1p suggested that the interactions of the isolated SH3Cyk3 with Inn1p and

Nba1p and of SH3Hof1 with Inn1p, Nba1p, and Cyk3p are principally strong enough

to recruit the respective complexes from their pre-mitotic localization to the site of

cytokinesis (Figures 2A, 2C, 3A).

Sho1p co-localizes with its ligands at the site of cytokinesis

Live cell imaging showed that Sho1-GFP appeared at the site of septum formation

shortly after the splitting of the septin ring (Figure 4B). Time-lapse studies of cells

co-expressing Sho1-CHERRY together with the GFP-fusions of either Hof1p, Inn1p,

or Cyk3p established that Sho1p reached the site of cell separation after Hof1p and

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Inn1p and shortly after or nearly at the same time as Cyk3p (Figure 4B). While the

ring characterized by Inn1p and Hof1p staining contracted during cytokinesis, Sho1p

and Cyk3p appeared more static (Figure 4B). Both proteins remained at the opposing

membranes of mother and daughter cells some time after cell separation (Figure 4B).

These data and the work by others suggest the following temporal sequence of

assembly and disassembly of the HICS-complex at the site of cell division: Hof1p,

Inn1p, Cyk3p, Sho1p – Inn1p, Hof1p, Cyk3p, Sho1p (Lippincott and Li, 1998;

Meitinger et al. 2010, 2010; Nishihama et al., 2009).

Taken together, our protein interaction maps and the timing of protein appearances at

the bud neck predict that the specific localization of Cyk3p and Inn1p during

cytokinesis depends on Hof1p but not on Sho1p (Figure 3C, 4B). Indeed, the single

deletion of Sho1p had only a minor effect on the localization of Inn1-GFP and

increased the fraction of cells containing Cyk3-GFP at the bud neck (Table S3). The

absence of Hof1p reduced Inn1p and Cyk3p at the bud neck quite dramatically (Table

S3). In comparison, the localization of Sho1p during cytokinesis did not depend on

the presence of Hof1p (Table S3).

Sho1p functionally contributes to cytokinesis

The high connectivity of Sho1p within this cytokinesis interaction network suggests a

unique role of Sho1p in this biological process. Electron microscopy of Δsho1 cells

revealed a large fraction of cells displaying a normally structured primary and

secondary septum. The primary septa of a minority of the cells displayed additional

invaginations of the plasma membrane close to the site of septum formation (Figure

5A, and Figure S2). In some cells the ingression of the plasma membrane during

cytokinesis appeared asymmetrically (Figure S2). More prominently, half of the

inspected cells displayed a secondary septum approximately twice as thick as seen in

wild type cells. Occasionally, we observed lacunae of cytosol within this enlarged

secondary septum (Figure 5A, B).

What are the specific functions of Sho1p and its interaction states within the above-

established cytokinesis network? Our results suggest that Hof1p and Sho1p can

compete for binding to the C-terminal proline-rich motifs in Inn1p and Cyk3p and

might thus perform similar or even redundant functions within this network (Figures

1-3). To address this question we sporulated a diploid Δhof1;Δsho1 strain. Spores

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harboring the double deletion could be recovered but appeared less frequently than

would have been expected for non-interacting genes (Table S2, Figure 6F). The

diameters of the recovered Δhof1;Δsho1 colonies were roughly half in size compared

to the Δhof1 colonies (Table S2). We repeated the sporulation in the presence of a

HOF1-expressing centromeric plasmid and tested the ability of Sho1p and a mutant

lacking the SH3 domain (sho11-240) to replace the plasmid-borne Hof1p in the

Δhof1;Δsho1 strain. Sho1-MYC partially complemented the growth defect of the

strain carrying the double deletion (Figure 6A). The complementation disappeared

under conditions of low Sho1-MYC expression (Figure 6A). Sho11-240 did not

complement the double deletion and instead seemed to exert a negative effect on the

growth of the cells (Figure 6A, F). This allele-specific enhancement was confirmed

by disrupting the reading frame of the genomic SHO1 before the start of its C-

terminal SH3 domain. Cells lacking HOF1 and expressing this allele instead of the

full length SHO1 were not viable (Figure 6B). The loss of the SH3Sho1 was, however,

tolerated in cells lacking MYO1 or CHS2 (Figure S3).

To further test the significance of the interactions between Sho1p and its targets

during cytokinesis we overexpressed SH3Sho1 as a cytosolic CHERRY-SH3Sho1 fusion

or as a membrane-anchored SH3Sho1-Sso1p fusion in wild type cells and cells lacking

HOF1. The expression of SH3Sho1-Sso1p specifically impaired the growth of Δhof1

cells, implying that the interactions between Sho1p and its targets become important

for viability under these conditions (Figure 6C).

Hof1p is the major determinant for Inn1p- and Cyk3p-localization at the bud neck

(Table S3). To estimate the contribution of Sho1p we compared the localization of

both proteins in cells lacking HOF1 and SHO1 with those lacking only HOF1. Table

S3 shows that the deletion of Sho1p further impaired the correct assembly of Cyk3p

and Inn1p at the division site.

Sho1p contacts Inn1p directly at the P4 motif and indirectly at the P2 motif via

Cyk3p (Figure 3C). To dissect the importance of either site for the cytokinetic

function of Sho1p we created an allele of INN1 (inn11-211) that lacked the two C-

terminal proline-rich motifs including the direct binding sites to Hof1p and Sho1p

(Figure 3C). This allele keeps the physical interaction with Sho1p or Hof1p indirectly

via Cyk3p. Sporulation of a heterozygous inn11-211;Δsho1 strain yielded haploid

inn11-211, Δsho1, and inn11-211;Δsho1 cells. Compared to the wild type a slightly

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reduced diameter of the colonies of the inn11-211 spores reflected the influence of the

C-terminal interaction sites on the activity of Inn1p (Table S2). The cells were

affected by a strong defect in cytokinesis (Figure S3). The further reduced colony-

diameters of the inn11-211;Δsho1 spores demonstrated a negative genetic interaction

between inn11-211 and SHO1 (Table S2, Figure 3D). This finding supports the

functional importance of Sho1p for cytokinesis by either recruiting Inn1p to the

membrane and /or by providing a complementary, Inn1p-independent pathway of

cytokinesis. To distinguish between these two alternatives we sporulated a

heterozygous inn11-211;Δcyk3 diploid and found no obvious genetic interaction

between this specific inn1 allele and CYK3 (Table S2). As Cyk3p is necessary to link

Inn11-211 to Sho1p this result argues for an additional Inn1p-independent function of

Sho1p in cytokinesis (Figure 3D).

The Nub-array identified Nba1p as interaction partner of the SH3 domains of Hof1p

and Cyk3p (Tonikian et al., 2009). An Inn1p-like scaffold function of Nba1p could

however be ruled out by our observation that the deletion of NBA1 does not impair

the growth of cells expressing the inn11-211 allele (Table S2).

The physical connections between Cyk3p, Hof1p, and Sho1p are relevant for

cytokinesis.

The simultaneous deletion of HOF1 and CYK3 is lethal for the cell (Korinek et al.,

2000). To evaluate the functional significance of the interaction of Cyk3p with

SH3Sho1 and SH3Hof1 we tested different alleles of CYK3 for their abilities to rescue

the growth of a Δhof1;Δcyk3 strain (Figure 6D, F). A CYK3 allele lacking the SH3

domain-encoding sequence (cyk3119-885) was not functional in this genetic background

(Figure 6D). The N-terminal fragment of Cyk3p still harboring the SH3 domain and

the proline-rich motif (Cyk31-194-GFP) retained a residual Cyk3p activity when

overexpressed (Figure 6D). Importantly, a CYK3 allele (cyk3ΔP-MYC) where the

contact site to Hof1p and Sho1 was destroyed rescued the Δhof1;Δcyk3 strain poorly

and only upon over-expression (Figure 6D, F). This very reduced ability of Cyk3ΔP-

MYC to functionally replace Cyk3p is not due to an intrinsic instability of the protein

as it is expressed at similar levels as the wild type protein (Figure 6E). The

experiments assign SH3Cyk3 an active role during cytokinesis that is supported by the

C-terminal tail domain and the ability of the P1 motif to interact with Hof1p or Sho1p

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(Figure 6F). The binding partner of this C-terminal domain, Slt2p, was already

discussed to activate Chs2p (Lesage et al., 2004; Lesage et al., 2005; Tong et al.,

2004). We thus propose that independently of Inn1p, Cyk3p might recruit the MAP

kinase Slt2p to modulate the enzymes responsible for primary and/or secondary

septum formation through phosphorylation. The observation that a SLT2 deletion is

not tolerated in Δmyo1 but in Δchs2 cells further supports this hypothesis (Rodriguez-

Quinones and Rodriguez-Medina, 2009; Yoshida et al., 2009) .

The synthetic lethality between HOF1 and CYK3 additionally suggests that either of

the two proteins acts in separate, partially complementing branches of cytokinesis

(Tolliday et al., 2001). Through our interaction analysis we were able to define with

P1 and the C-terminal domain two further separable regions within Cyk3p besides the

N-terminal SH3-domain (Figure 2). As none of both Cyk3p fragments complemented

the Δhof1;Δcyk3 strain we can exclude that either of them alone contributes

significantly to this alternative pathway (Figure 6D). Hof1p is a multi-domain protein

(Figure 1D). We tested different fragments for their ability to complement the

function of Hof1p in the absence of Cyk3p. Fragments missing the FCH, the F-Bar,

or the region linking the F-Bar with the SH3 domain failed to complement, whereas a

hof11-609 allele only lacking the SH3 domain fully rescued the Δhof1;Δcyk3 double

deletion (Figure 7A). The outcome of these experiments suggests that the SH3-

domains of Cyk3p, Sho1p, and Hof1p collaborate in the same pathway, whereas the

more N-terminal domains of Hof1p are additionally involved in at least one

alternative pathway. To reveal whether Sho1p contributes to this alternative path we

repeated the experiments in a strain lacking CYK3, HOF1, and SHO1, but harboring

an extra-chromosomal copy of HOF1. Independently of the introduced alleles, the

triple deletion grew less well than the corresponding Δhof1;Δcyk3 double deletion

(Figure 7B). hof11-609 complemented the wild type allele in the triple knockout less

efficiently than in the double deletion (Figure 7B). The reintroduction of CYK3 under

control of the PMET17 promoter allowed us to compare the growth of the Δhof1 with

the Δhof1;Δsho1 cells under different Cyk3p expression levels. Increasing the

expression of Cyk3p partially compensated for the single loss of HOF1 or the

simultaneous deletions of HOF1 and SHO1. Taken together, these experiments

demonstrate a negative genetic interaction between SHO1 and CYK3 and indicate that

the SH3 domains of Hof1p and Sho1p operate upstream of Cyk3p (Figure 7C).

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Discussion

The aim of this study is to better understand the complex interplay among the

proteins of the cytokinesis machinery. We complemented our protein interaction

analysis by measuring genetic interactions between alleles of members of the

established network that lacked defined protein interaction sites for their binding

partners. Following this experimental strategy supported by microscopic studies we

could define the osmosensor Sho1p as a novel member of the cytokinesis network. Its

contribution to cytokinesis depends on its C-terminal SH3 domain that is responsible

for interactions with the proteins Hof1p, Inn1p, and Cyk3p. Sho1p is the only

member of the herein defined HICS-complex that is an integral membrane protein

and thus permanently embedded in the cell membrane. Via interaction with Hof1p

and Inn1p, Sho1p may present a link between the AMR and the plasma membrane

during cytokinesis (Figure 8A). As the phenotype of a SHO1 deletion is mild

compared to the phenotype of a MYO1 deletion it is highly likely that Sho1p acts with

other proteins to fulfill this function. Potential candidates are again Hof1p and Inn1p,

as both genes display negative genetic interactions with SHO1 (Table S2).

Our data collectively suggest that the role of Sho1p in cytokinesis is not linked to its

function as an osmosensor. The functional consequences of its interactions with the

cytokinetic proteins thus differ from its reported interaction with Fus1p during

mating. Here, the interaction interrupts the MAP-kinase pathway of the high-

osmolarity response to increase the cells’ tolerance to the conditions that occur during

cell-cell fusion (Nelson et al., 2004).

By including Sho1p we can upgrade our knowledge on the choreography of events

during cytokinesis (Jendretzki et al., 2009; Lippincott and Li, 1998; Meitinger et al.,

2011; Nishihama et al., 2009). Hof1p assembles as a double ring at the bud neck of

large budded cells. In late anaphase Hof1p relocalizes to a single ring between the

split septins. This event is accompanied by the phosphorylation of the central region

of Hof1p by Dbf2p (Meitinger et al., 2011). Inn1p and later Cyk3p assemble with

Hof1p at that central ring to from a AMR-Hof1p-Inn1p-Cyk3p complex (Meitinger et

al., 2011; Nishihama et al., 2009). The delivery of Sho1p into the region between the

split septin rings during anaphase increases its effective concentration, thereby

facilitating the binding of SH3Sho1 to the proline-rich region within the RLS of Hof1p

and the proline-rich motifs of Inn1p and Cyk3p to finally form the HICS-complex

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(Figures 4B, 8). Reflecting their different roles during membrane contraction and

septum synthesis, the subunits of the HICS-complex display extensive negative

genetic interactions among each other. One may speculate that mediating different

yet related activities via one multifunctional complex might allow their extremely

precise and efficient coordination in time and space (Figures 7D, 8A) (Nishihama et

al., 2009; Sanchez-Diaz et al., 2008).

The HICS-complex contains with Cyk3p, Hof1, and Sho1p three SH3-domain-

containing proteins. Two, namely Cyk3p and Hof1p, serve also as SH3-acceptors and

all three bind with partially overlapping specificities to the scaffold protein Inn1p

(Figure 3C). These features increase the potential combinatorial diversity of this

interaction network through multiple protein interaction states and might equip the

HICS-complex with unique properties normally not encountered in protein

complexes. For example, a 1:1:1:1 stoichiometry of its members can assemble into

seven isomeric interaction states that only differ in the positions of the edges (Figure

8B). This rich molecular heterogeneity might find its counterpart in the fluctuations

of the relevant parameters during cytokinesis such as membrane-AMR distance,

curvature of the membrane, chemical composition of the membrane and of the AMR.

The HICS-complex might adjust to these changes by selecting the best fitting from its

spectrum of interaction states and accommodating varying combinations of further

interacting proteins (Figures 3C, 8B).

Sho1p has the potential to form homo-oligomers (Table S1) (Hao et al., 2007). Once

exceeding a critical concentration, these oligomers might condense the isolated

interaction states into a single net (Figure 8C). Due to the highly flexible

arrangements of interactions this net might display liquid-like properties that could

define a new compartment of cell-cell separation below the membrane (Brangwynne

et al., 2009; Hyman and Brangwynne, 2011). The structure might then serve as

attachment site for the AMR to couple AMR contraction to the ingression of the

plasma membrane. It is even conceivable that during the final steps of cytokinesis

this conformational flexibility might allow this structure to tether the two opposing

membranes for their fusion. The interaction of Sho1p with Sro7p, a stimulator of

exocytosis, as well as their negative genetic interaction supports this hypothesis

(Table S1) (Aguilar et al., 2010).

Material and Methods

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Growth conditions, yeast strains, and genetic methods

Standard culture media and standard yeast genetic methods were used as described

(Guthrie and Fink, 1991; Dünkler et al., 2012). Media for the Split-Ubiquitin

interaction assay or media to select for the loss of URA3-containing plasmids

contained 1 mg/ml 5-fluoroorotic acid (5-FOA). Yeast strains JD47, JD53, and JD51

are as described (Dünkler et al., 2012).

Construction of Nub- and Cub-fusion genes and other molecular manipulations

Construction of Nub- and Cub-fusion genes were essentially as described (Dünkler et

al., 2012; Hruby et al., 2011). Insertion of the PMET17 promoter, gene deletion and

epitope tagging were performed by PCR-based methods as described (Janke et al.,

2004; Knop et al., 1999). The GST- fusions were obtained by placing the ORF of the

respective gene or gene fragment behind the E. coli GST sequence on the pGEX-6P-1

vector (GE Healthcare, Freiburg, Germany). The AGT fusions were expressed from

the vector pAGT-express, a pet15b derivative, where the ORF of the respective gene

was inserted into a multi-cloning site located between the upstream 6XHIS-tag-

coding sequence and the downstream AGT-coding sequence.

Mutations in CYK3 were introduced by using primers annealing at the respective site

but carrying a mismatch to change the codon by the ensuing PCR. The mutated

sequence of Cyk3ΔP between position 185 and 192 reads ASLASLA instead of

PPLPPLP.

Split-Ub Interaction analysis

Large scale Split-Ubiquitin assays were performed as described (Dünkler et al., 2012

; Hruby et al., 2011). Measuring interactions between individual Nub- and Cub-fusion

proteins by spotting yeast cells expressing both fusions onto 5-FOA and SD Ura-

containing media was essentially as described (Eckert and Johnsson, 2003).

In vitro binding assays

GST-fusion sepharose slurries were incubated for 1 h at 4°C under rotation with

either 1 ml of yeast cell extract containing the MYC-tagged proteins or with E.coli

extracts containing AGT-fusion proteins. Beads with bound proteins were separated

by 2 min centrifugations at 6000 rpm and washed 4 times with PBS. The supernatant

was discarded and 100 μl elution buffer (50 mM TRIS pH 7.0 with 20 mM reduced

glutathione) was added, and gently mixed for 10 min at 4°C. Finally, beads were

again centrifuged for 2 min at 6000 rpm and the supernatant was transferred to a fresh

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tube, mixed with loading buffer (120 mM Tris-Hcl pH 6.8, 4 % (w/v) SDS, 20 %

(v/v) glycerine, 0.015 % (w/v) bromophenolblue, 100 mM DTT), boiled for 5 min.,

and analyzed by SDS-PAGE and immunoblotting. Immunoblotting was done as

described (Hruby et al., 2011).

Preparation of yeast cell extracts

For pull-down experiments, yeast cell cultures were grown to an OD600 of 1.5,

pelleted, washed once in ice-cold water, and then transferred into liquid nitrogen. The

cell pellets were ground in liquid nitrogen using a mortar. The cell powder was

collected in protein extraction buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 1 mM

EDTA) containing 1x protease inhibitor cocktail Complete (Roche Diagnostics,

Mannheim, Germany), 1 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl

fluoride (PMSF). 0.1% Triton X-100 was added, incubated for 10-20 min on ice, and

extracts were clarified by centrifugation.

Expression and immobilization of AGT- and GST-fusion proteins

E. coli cells (BL21, Amersham, Freiburg, Germany) expressing AGT- or GST-

fusions were grown at 37°C till an OD600 of 0.6, and the expression of the AGT- and

GST-fusion proteins were induced by 0.5 mM IPTG. The cells expressing the GST-

fusions were shaken for additional 4 h at 37°C, harvested by centrifugation, and

resuspended in PBS containing 1x protease inhibitor cocktail, 0.4 mM PMSF and 1

mg/ml lysozyme (Sigma-Aldrich Chemie, Steinheim, Germany). After incubation for

10 min at 4°C, cells were sonicated, and the lysates were clarified by centrifugation at

13,000 rpm for 15 min at 4°C, and either stored at –80°C or immediately incubated

with PBS-equilibrated glutathione-sepharose 4B beads (GE Healthcare, Freiburg,

Germany) for 1 h at 4°C under rotation. Finally, bound material was washed four

times in 1x PBS.

The cells expressing AGT-fusions were incubated under inducing conditions for 6 h

at 18°C. Extract preparation was as above but containing additional 0,5% Triton X-

100 in the lysis buffer.

Electron microscopy

Exponentially growing yeast cells were arrested with α-factor to a final concentration

of 5 μg/ml over 2-3h. After washing out the α-factor, the cells were released into the

fresh medium and allowed to proceed through one cell cycle. Samples for electron

microscopy were prepared according to Walther and Ziegler (Walther and Ziegler,

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2002) with some modifications. Cell-pellets were resuspended in fresh medium at

30°C and rapidly frozen on the Saphire glass surface in a Compact 01 high-pressure

freezer (M. Wohlwend GmbH, Sennwald, Switzerland). Samples were subjected for

freeze-substitution (with 0.2 % osmiumtetroxide, 0.1 % uranyl acetate, 5 % water in

acetone), washed with water free acetone, stepwise embedded in epon, polymerized

and cut with a Leica Ultracut UCT ultra-microtome. Ultrathin sections (about 70 nm)

were post-stained with uranyl acetate and observed with a Philips 400 transmission

electron microscope at an accelerating voltage of 80kV. Images were collected by a

CCD camera and analyzed with the EMMENU software and Photoshop.

Measurements of septum thickness were performed with ImageJ software.

Microscopy, live cell imaging.

Differential interference contrast (DIC) and fluorescence microscopy were performed

using the Delta Vision System (Applied Precision, Issaquah, USA). The System is

equipped with the Olympus IX71 microscope, with a 100x Plan-Apo/1.4 objective

lens and the charge-coupled-device (CCD) camera (Photometrics, München,

Germany). Images were acquired and analysed with the softWoRx software of the

Delta Vision System, and processed using Adobe Photoshop. Fluorescent proteins

were visualized using a live cell filter set (Chroma Technology Corp., Olching,

Germany) using the EGFP (λex470-λem525) and mCherry filter (λex572-λem632)

respectively.

Yeast strains were grown overnight in liquid selective media, diluted the next day,

and grown with or without methionine to middle log phase at the indicated

temperatures. Cells were fixed after washing once in selective media by incubation in

4% paraformaldehyde for 10 min at room temperature. Cells were washed twice in 1x

PBS before analysis.

For time-lapse microscopy exponentially grown cells were diluted to a density of

3x106 cells/ml and mounted on an agarose pad. Temperature was held constant at

30°C by a Delta Vision System supplied temperature chamber.

Acknowledgements

The work was supported by a DFG Research Grant to N.J. (JO 187/5-1). K.L. was

supported by a Marie Curie Research Training Network of the European Union

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(PROSA, 019475). We thank Dr. Rong Li for the plasmid pLP8. The authors declare

that they have no conflict of interest.

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Figure legends

Figure 1

Sho1p interacts with known members of the cytokinesis pathway. (A) Split-Ub

interaction assay of yeast strains co-expressing Sho1CRU (left panel) or Sho11-

297CRU (right panel) with the selected Nub-fusion proteins. Cells were grown to an

OD600 of 1 and 4 μl of the cultures spotted as ten-fold serial dilutions on 5-FOA

plates. Nub without a C-terminally attached ORF (Nub-) serves as a negative control in

this and the following Split-Ub assays. Growth of the cells indicates interactions

between the co-expressed Ub-fusions. Note that the Sho11-297CRU-expressing cells

show already some growth even in the presence of non-interacting Nub-fusions. Here,

only a significantly elevated growth serves as indicator for interaction. (B) Protein

extracts of yeast cells expressing MYC-taged Pbs2p (lanes 1-3), Hof1p (lanes 4-6),

Cyk3p (lanes 7-9), and Inn1p (lanes 10-12) were treated with bacterially expressed

GST (lanes 2, 5, 8, 11) or GST-SH3Sho1 bound to sepharose beads (lanes 3, 6, 9, 12).

Glutathione eluates (lanes 2, 3, 5,6, 8, 9, 11, 12) and yeast extracts (lanes 1, 4, 7, 10)

were separated by SDS-PAGE followed by immunoblotting with anti-MYC

antibodies. (C) Bacterially expressed AGT (lane 3), AGT-fusions of Cyk3160-210

(lanes 1, 4) and its proline-rich motif-missing mutant (Cyk3160-210ΔP-AGT; lanes 2, 5)

were treated with GST- (lanes 1, 2) or GST-SH3Sho1-bound beads (lanes 3-5).

Glutathione-eluates were separated by SDS-PAGE followed by immunoblotting with

anti-AGT antibodies. (D) Upper panel: cartoon of Hof1p indicating the FCH and SH3

domain as well as the proline-rich motifs at position 333-338 (P1) and 386-392 (P2).

Lower panel: as in (C) except that Hof1269-349-AGT (lanes 1-3) and Hof1366-423-AGT

were precipitated with GST- (lanes 2, 5) or GST-SH3Sho1-bound beads (lanes 3, 6).

Bacterially expressed AGT-fusions of Hof1269-349 and Hof1366-423 are shown in lane 1,

and 4. (E) As in (C) except that Inn1131-211-AGT (lanes 1-3), Inn1291-341-AGT (lanes 4-

6), and Inn1351-409-AGT (lanes 7-9) were precipitated with GST- (lanes 2, 5, 8) or

GST-SH3Sho1-bound beads (lanes 3, 6, 9). Bacterially expressed AGT-fusions of

Inn1131-211, Inn1291-341, and Inn1351-409 are shown in lanes 1, 4, and 7. (F) As in (C)

except that Msb11-39-AGT (lanes 1-3) was precipitated with GST- (lane 2) or GST-

SH3Sho1-bound beads (lane 1). Bacterially expressed and Anti-AGT-probed Msb11-39-

AGT is shown in lane 3. (G) Summary of interaction data: The proline-rich motifs

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(white nodes) of the proteins Pbs2p, Hof1p, Cyk3p, Inn1p, and Msb1p might

compete for binding (edges) to the SH3 domain (grey node) of Sho1p.

Figure 2

Dissection of Cyk3p-interaction sites. (A) Upper panels: Split-Ub interaction assays

of Cyk3p and truncated versions of Cyk3p as in (1A). Lower panel: Cartoon of

Cyk3p showing the N-terminal SH3 domain, the proline-rich motif between residues

183 and 191, and the Trans-Glutaminase-Domain between residues 500 and 600. (B)

Cells co-expressing Cyk3119-885CRU lacking the SH3 domain or the same truncation

of Cyk3p with a mutated the proline-rich motif (Cyk3119-885ΔPCRU), and Nub-fusions

of the identified Cyk3p-ligands were spotted in ten-fold serial dilutions on media

containing 5-FOA. Nub-Guk1p co-expressing cells served as an additional negative

control. (C) Yeast cells containing the C-terminal truncations of Cyk3p still harboring

the SH3 domain and the proline-rich motif (Cyk31-267CRU) or the same truncation of

Cyk3p with mutated proline-rich motif (Cyk31-267ΔPCRU) and co-expressing Nub-

fusions of Hof1p or its fragments (see 1D) were spotted on 5-FOA containing media.

Expression of Nub-fusions were induced by addition of copper to 50 mM, except Nub-

SH3Hof1, that showed even under non-inducing conditions high activity. (D) Pull-

down assay as in 1C, except that AGT (lanes 1, 4), Cyk3160-210-AGT (lanes 2, 5) and

Cyk3160-210ΔP-AGT (lanes 3, 6) were incubated with immobilized GST-SH3Hof1. Anti-

AGT-probed Gluthatione-eluates of the beads are shown in lanes 1-3 and extracts

before incubation are shown in lanes 4-6. (E) Summary of interaction data.

Horizontal lines connect SH3-domains (grey nodes) with proline-rich stretches (white

nodes) or other regions within one protein, whereas all other edges indicate protein

interactions between different proteins (black nodes) or their domains.

Figure 3

Dissection of Inn1p- and Hof1p interaction sites. (A) Split-Ub interaction assay as in

(1C) with Hof1CRU and a C-terminal fragment of Hof1p lacking the N-terminal

FCH domain and the P1 motif (Hof1343-669CRU). Split-Ub interaction assay of

Inn1CRU or fragments of Inn1p containing different combinations of the proline-rich

motifs P1-P4 (see 3C). (C) Upper panel: Summary of interaction data as in 2E. Lower

panel: Cartoon of Inn1p indicating the N-terminal potential lipid-binding C2-domain

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and the four proline-rich motifs. (D) Protein interaction states as in 3C but lacking P3

and P4 of Inn1p and either Cyk3p, or Sho1p. / indicates no, and – indicates a

significant negative genetic interaction (GI).

Figure 4

(A) Split-Ub interaction assay as in (1A) between the SH3 domain of Sho1p

(SH3Sho1CRU) and its known interaction partners. (B) Fluorescence microscopy of

cells co-expressing Sho1-GFP and Cdc11-CHERRY, Sho1-CHERRY and Hof1-GFP,

Sho1-CHERRY and Inn1-GFP, and Sho1-CHERRY and Cyk3-GFP. Except Cdc11-

CHERRY all fusions were expressed under control of the PMET17 promoter from their

chromosomal locations. Arrowheads indicate the first appearance of Sho1p, Cyk3p or

Inn1p at the bud neck, or the double ring of Cdc11p and Hof1p at the bud neck. The

Length of the white bars indicates 5 mm.

Figure 5

Electron microscopy of wild type (a-c) and Δsho1 cells (d-i). Arrowheads indicate

septa between the cells, double arrowheads indicate the plasma membrane. Cytosol-

enclosed lacuna (LA), cell wall (CW). (B) Distribution of cell wall diameters at the

division sites of separating wild type and Δsho1 cells.

Figure 6

Genetic interactions. (A) Δhof1;Δsho1 cells carrying a plasmid expressing HOF1 and

URA3 were transformed with plasmids expressing the indicated genes under control

of the PMET17 promoter. 4 μl of cultures of 1 OD600 were spotted in tenfold serial

dilutions on media containing uracil, 5-FOA, and the indicated concentrations of

methionine to repress (1mM) or modestly induce (70 μM) the PMET17 promoter. (B)

Cells expressing the sho11-240 allele and HOF1 on a URA3-containing plasmid in wild

type or Δhof1 cells were grown on media supplemented with uracil or uracil and 5-

FOA. Wild type and Δhof1 cells served as controls. (C) Wild type and Δhof1 cells

were transformed with the indicated constructs under control of the PMET17-promoter

and spotted in tenfold serial dilutions on media selecting for the presence of the

plasmids and containing no methionine to fully induce the expression of the gene

fusions. Cells were grown for four days at 34oC. (D) As in (A), except that the

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Δhof1;Δcyk3 cells expressing HOF1 and URA3 from the same plasmid contained

different fusion alleles of CYK3 under control of the PMET17 promoter. Cells were

grown on medium containing 0 or 70 μM of methionine to fully or modestly induce

the expression of the fusion genes. (E) Anti-MYC immunoblot of extracts originating

from cells expressing MYC-labeled CYK3 (lane 2), a mutant of CYK3 lacking the

proline-rich stretch between residues 183 and 191 (lane 3), or the empty PRS315

plasmid (lane 1). (F) Visualization of protein networks as in Figure 3D. – indicates a

negative genetic interaction (GI) between the respective alleles.

Figure 7

Functional dissection of the CYK3-dependent pathway. (A) Δhof1;Δcyk3 cells

carrying a plasmid expressing CYK3 and URA3 were transformed with plasmids

expressing the indicated alleles of HOF1 under control of the PMET17 promoter or an

empty vector. 4 μl of cultures of 3 OD600 were spotted in tenfold serial dilutions on

media containing uracil, 5-FOA, and the indicated concentrations of methionine to

modestly (70 μM) or fully (0 M) induce the PMET17 promoter. Growth was monitored

after 4 days (B) Δhof1;Δcyk3;Δsho1 cells carrying a plasmid expressing HOF1 and

URA3 were transformed with plasmids expressing the indicated HOF1 alleles and

analyzed as in (A), except that cells were grown on media containing 1 mM Met for 6

days. (D) As in (B), except that the indicated cells expressed low, moderate and high

levels of CYK3-MYC (1mM, 70 μM, 0 M Met). (D) Information flow within the

Cyk3p-dependent pathway of cytokinesis. Protein interactions are indicated with

black, and functional activities with red arrows. Hof1p and Sho1p recruit and activate

Cyk3p via their SH3 domains. Cyk3p activates Inn1p via its SH3 domain, and at the

same time recruits Slt2p and potentially other factors (Z) to the site of septum

formation. Inn1p (either directly or via factor Y) and Slt2p might coordinate primary

and secondary septum formation during cytokinesis. Both, Hof1p and Sho1p, perform

additional functions during cytokinesis outside the Cyk3p-dependent pathway.

Figure 8

Models for the organization of the HICS-complex. (A) The HICS-complex connects

the membrane with the AMR. Interactions of Hof1p and Inn1p with the membrane

are still hypothetical and the interactions of both with the AMR not yet understood.

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Shown is a HICS-complex with a 1:1:1:2 stoichiometry. Other configurations are

equally likely but not shown. (B) The seven isomeric interaction states of the HICS-

complex with a 1:1:1:1 stoichiometry. (C) A high concentration of Sho1p leads to

Sho1p oligomerization that condenses the isolated interaction states into a net of

proteins with a highly flexible arrangement of interactions.

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