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The Budding Yeast PCH/F-BAR Proteins

and .

Author Information

* and **.

* Corresponding Authors: Alan L. Munn—School of Medical Science, Griffuth University, Southport, Queensland, 4222, Australia. Email: ua.ude.htffirg@nnum.a
** Barbara A.T. Winsor—UMR7156, Center National de Recherche Scientifque, Universite Louis Pasteur, Strasbourg, 67084, France. Email: rf.gbsarts-u.cmbi@rosniw.b.

The budding yeast Saccharomyces cerevisiae genome encodes two classical Pombe Cdc15 Homology (PCH) proteins: Hof1p (or Cyk2p) and Bzz1p (or Lsb7p). Like mammalian PCH proteins, both have an N-terminal F-BAR domain and C-terminal Src Homology 3 (SH3) domain(s). The yeast genome also encodes two proteins that possess N-terminal F-BAR domains but have a C-terminal Rho GTPase activating protein (GAP) domain instead of an SH3 domain: Rgd1p and Rgd2p. Hof1p level is regulated during the cell cycle with peak expression at late anaphase/telophase. Hof1p localizes to rings at the site of cytokinesis. Two pathways of cytokinesis in S. cerevisiae differ in their dependency on the contractile actomyosin ring. Loss of Hof1p appears to block the actomyosin-ring-independent pathway of cytokinesis. The SH3 domain of Hof1p is dispensable for localization and cytokinesis, but negatively regulates Hof1p localization and both cytokinesis pathways, apparently as part of an important regulatory switch. Bzz1p is found throughout the cell cycle and localizes to dynamic cortical patches that contain actin filaments at sites of polarized surface growth. Loss of Bzz1p has no obvious phenotype, but Bzz1p deficiencies when combined with other mutations and in vitro polymerization assays, show a role for Bzz1p through its SH3 domains in actin filament assembly and endocytosis. Rgd1p and Rgd2p are GAPs for Rho family GTPases that negatively regulate different aspects of polarized cell growth including the actin cytoskeleton, directed secretion and cell wall remodelling. Whether the F-BAR domains of these proteins bind and bend membranes like those of mammalian PCH proteins has not yet been explored.

Introduction to Saccharomyces cerevisiae

S. cerevisiae is a unicellular and free-living member of the Fungi Kingdom. Unlike mammalian cells, S. cerevisiae cells are enclosed in a thick cell wall composed of polysaccharide. Another distinguishing feature from mammalian cells and from fission yeast Schizosaccharomyces pombe cells is that they divide by budding, an asymmetrical process in which a daughter cell emerges and is subsequently released from a site on the mother cell surface. Like other eukaryotic cells, S. cerevisiae cells have internal membrane compartments including a nucleus, endoplasmic reticulum (ER) (which is contiguous with the nuclear envelope), Golgi, mitochondria, peroxisomes and the vacuole (a hydrolytic compartment equivalent to the mammalian lysosome). Tey proliferate by undergoing multiple rounds of a cell division cycle that includes chromosome replication, chromosome segregation/nuclear division and finally cytoplasmic partitioning into two progeny cells.1 The six Rho GTPases in S. cerevisiae, Cdc42p and Rho1-5p play a central role in regulating polarized growth processes including budding, septin organization, secretion and the stimulation of cell wall synthesis.2-6

In S. cerevisiae it is possible to judge the stage of the cell division cycle of any particular cell by its morphology. Cells in late G1 phase of the cell cycle have no bud. Upon entry into S phase, a bud starts to emerge from the surface of the mother cell. Troughout S phase, the bud grows rapidly at its tip. In G2 phase the bud switches from polarized tip growth to isotropic growth and becomes ovoid in shape. In mitosis (M), the bud approaches the size of the mother cell and the process of nuclear division provides the bud with a nucleus. Upon exit from M phase, the cells undergo cytokinesis, the process by which the cytoplasmic contents of the mother and daughter cells are divided following the completion of nuclear division.1,7,8

Septins are important proteins in polarity. The position of the bud neck in S. cerevisiae is determined by septins. Tey are believed to comprise 10 nm diameter filaments seen to line the bud neck in electron micrographs. Septins are the first proteins to assemble at the site on the mother cell where the bud will emerge. Tey form a small ring at this site and the new bud grows out from within the septin ring. During M phase when the bud approaches the size of the mother cell the single septin ring splits and separates to form two rings, one on the mother cell side and one on the daughter cell side of the neck. During cytokinesis the two septin rings move further apart. The mother and daughter cell each inherit one septin ring. Afer cell separation the septin ring becomes diffuse and the septins appear to migrate around the cortex and concentrate at the new bud site.1,7,9

The Saccharomyces cerevisiae Actin Cytoskeleton

Like mammalian cells S. cerevisiae cells have both actin-based microflament and tubulin-based microtubule cytoskeletons and many components are equivalent. There are three major F (filamentous)-actin structures in S. cerevisiae: cortical actin patches, cytoplasmic actin cables, the cytokinetic actomyosin ring. Cortical actin patches are highly dynamic and short-lived spots of F-actin that continually form at the cortex, move very rapidly over short distances and then disassemble. Cortical actin patches are made up of highly branched actin filaments nucleated by a seven-subunit protein complex called the Arp2/3 complex. The Arp2/3 complex has weak activity unless stimulated by a Nucleation Promoting Factor (NPF) that in S. cerevisiae include Abp1p, Pan1p, the type I myosins (Myo3p and Myo5p) and Las17p.1,10-12 Las17p is the unique yeast ortholog of the mammalian Wiskott-Aldrich Syndrome Protein (WASP) family and the only activator to bind monomeric actin. Las17p incorporates signals from upstream Rho family GTPases (Cdc42p, Rho3p and Rho4p) and interacts with a number of proteins including verprolin (Vrp1p) (the human WASP-Interacting Protein, or WIP, homolog) and the type I myosins in a module that signals to the Arp2/3 nucleator complex.13-15 CDC42 activates the Arp2/3 complex indirectly in mammalian cells by alleviating auto-inhibition in WASP and its ubiquitously expressed homolog N- (neuronal) WASP. In contrast, other WASP-related proteins such as mammalian WAVE (WASP-family Verprolin-homologous protein) and S. cerevisiae Las17p do not have GTPase binding domains. Las17p does not appear to be auto-inhibited, at least in vitro.16

Cytoplasmic actin cables are long F-actin filamentous structures that lie under the cell cortex. Actin cables are more long-lived than patches and serve as tracks for movement of transport vesicles and organelles (e.g., mitochondria). Actin cables are made up of linear actin filaments whose assembly is nucleated by two formins, Bni1p and Bnr1p.1,10-12,17-20

Cortical actin patches and actin cables are present in every cell in an asynchronous population. However, their distribution within the cell changes during the cell cycle. In late G1 actin patches concentrate at the presumptive bud site on the mother cell. Actin cables orient such that their tips also concentrate at the site of bud emergence. As the bud emerges and undergoes polarized growth in S phase the cortical actin patches concentrate in the bud and in particular at the fast growing tip. The actin cables orient along the mother cell-bud axis with their ends near the bud tip. In G2 the actin patches remain concentrated in the bud but are spread out rather than clustered at the bud tip. At this time the bud switches from polarized growth to isotropic growth and expands laterally to form an ovoid shape. Upon entry into mitosis actin patches and cables become randomly distributed. Finally, as cells exit mitosis actin patches repolarize to the bud neck and actin cables realign with their ends focussed towards the bud neck for cytokinesis.1,12,17,18

Cytokinesis in Saccharomyces cerevisiae

A cytokinetic actomyosin ring is an F-actin structure unique to dividing cells. The actomyosin ring is not composed of patches but rather has an even and continuous appearance. A key component of the actomyosin ring is the type II myosin, Myo1p. Myo1p assembles into a myosin ring in late G1 and this process requires septins. Actin filaments assemble in this ring to form an actomyosin ring in late M phase. The actin filaments that comprise the actomyosin ring are linear and their assembly is dependent on the formins Bni1p and Bnr1p but not on the Arp2/3 complex. Upon exit from M phase the actomyosin ring appears to constrict to a small dot and then disassembles. This constriction event coincides with septum deposition and partitioning of the cytoplasm.8,21,22

Cytokinesis in S. cerevisiae involves the deposition of a primary septum made of chitin. Chitin starts to be deposited on the mother cell side of the bud neck as early as G2, but the primary septum is only synthesized in late anaphase/telophase, coincident with actomyosin ring constriction. This primary septum initiates symmetrically at either side at the cell periphery and as it grows inward it divides the cytoplasm of the cell until it meets in the middle, thus giving rise to separate mother and daughter cells. Deposition of the primary septum is closely followed by deposition of a secondary septum that contains some chitin but is predominantly glucan like the rest of the cell wall. The primary septum is then digested by chitinases, resulting in cell separation.1,23

Budding Yeast PCH Proteins

The classical PCH proteins of yeast have been reviewed.24 However, knowledge of the PCH proteins of yeast has expanded tremendously since this review and it is timely to review this topic again.

The S. cerevisiae genome encodes two classical Pombe Cdc15 Homology (PCH) proteins: Hof1p (also known as Cyk2p) and Bzz1p (also known as Lsb7p). Hof1p and Bzz1p have a domain structure that closely resembles that of the mammalian PCH proteins. The N-terminal region of both proteins comprises a Fes/Cip4 Homology (FCH) domain predicted to be largely α-helical and to engage in coiled-coil interactions. Following the FCH domain of each protein is another coiled-coil domain (cc2). Together, the FCH and cc2 domains comprise the F-BAR domain (Cdc15 Homology domain). Hof1p and Bzz1p also have one or more PEST motifs, rich in the amino acids proline (P), glutamic acid (E), serine (S) and threonine (T). PEST motifs typically confer ubiquitination and proteasomal degradation on proteins that possess them. The C-terminus of both yeast PCH proteins feature Src Homology 3 (SH3) domains that bind target sequences containing proline (typically PXPXXP). Hof1p has a single SH3 domain while Bzz1p has two SH3 domains24 (Fig. 1). Despite their similar domain organization, Hof1p and Bzz1p differ in subcellular localization, protein interactions and mutant phenotypes and therefore fullfil distinct cellular roles. The budding yeast genome also encodes two additional proteins with N-terminal F-BAR domains with a Rho-GAP domain and no SH3 domain: Rgd1p and Rgd2p (Fig. 1).

Figure 1. Domain structure of the yeast PCH proteins.

Figure 1

Domain structure of the yeast PCH proteins. Shown are schematics that depict the domain organization of Hof1p, Bzz1p, Rgd1p and Rgd2p. Abbreviations: FCH: Fes/CIP4 Homology; PEST: sequence rich in proline (P) glutamic acid (E) serine (S) and threonine (more...)

In nomenclature convention for budding yeast wild type gene names are designated with a three-letter name in capital letters and italics, e.g., your favorite gene is YFG1. Recessive mutations are shown using lowercase letters and italics, e.g., yfg1. A gene deletion is designated with a delta, e.g., yfg1Δ. The protein is designated with only the first letter capitalized and the suffix “p” (e.g., Yfg1p).

Hof1p/Cyk2p

The HOF1 (Homologue Of Fifteen 1) gene was so-named because it encodes the first budding yeast homologue of S. pombe Cdc15.25 It is also referred to in the earlier literature as CYK2 (CYtoKinesis 2).26

Regulation of Hof1p expression

Endogenous Hof1p levels are cell cycle regulated.27,28 Hof1p is not detected in G1 and S-phase but becomes detectable upon passage through the G2/M boundary and peaks in anaphase/telophase. The mechanisms that govern the cell-cycle dependency of Hof1p levels are now emerging. First, HOF1 expression is subject to transcriptional regulation during the cell cycle. HOF1 transcription initiates in S-phase, steadily increases through G2/M, peaks in anaphase, rapidly declines after exit from mitosis and is undetectable in G1 phase.29 Placing the HOF1 gene under the control of a constitutive promoter causes Hof1p to be produced somewhat earlier (S-phase), but not in G1. This indicates that transcriptional regulation only partially accounts for the absence of Hof1p in G1.28 Hof1p levels are also regulated by ubiquitin-dependent proteolysis. Hof1p is degraded with a half-life of 55 min in asynchronous cells. The Hof1p PEST motif (residues 418-438) is critical for Hof1p proteolysis as deletion of the PEST motif increases Hof1p half-life to >120 min and results in detectable Hof1p levels in G1. The SCF (Skp1p/Cullin/F-box) E3 ubiquitin ligase mediates Hof1p degradation. There are several subtypes of SCF whose substrate specifcity is conferred by the F-box component. Hof1p degradation requires the F-box protein Grr1p as loss of Grr1p extends Hof1p half-life to >120 min. The PEST motif mediates Hof1p binding to the Grr1p F-box domain.28,30

Hof1p Subcellular Localization

Perhaps the most important clue to Hof1p function came from its subcellular localization. Hof1p forms rings positioned at or close to the bud neck. Consistent with the cell-cycle regulation of Hof1p expression, Hof1p rings are not apparent in G1 cells. However, in cells that have passed the G2/M boundary Hof1p localizes to a large ring on the mother cell side of the bud neck and colocalizes with one septin ring (Fig. 2). In M phase Hof1p is present in rings on both mother and daughter sides of the bud neck and colocalizes with the septin rings. In late anaphase or exit from mitosis (telophase) Hof1p localizes to a single ring that colocalizes with the contractile actomyosin ring (Fig. 2). Afer cell separation, Hof1p rings disappear and this corresponds to a dramatic decline in Hof1p protein levels. Proteolysis of Hof1p may occur at the bud neck. The Hof1p degradation factor Grr1p localizes to the bud neck in M-phase and interacts with Hof1p just prior to Hof1p proteolysis.24-28,31-36

Figure 2. Localization of Hof1p to rings at the bud neck.

Figure 2

Localization of Hof1p to rings at the bud neck. Shown are time-lapse images of cells expressing a Hof1pgreen fuorescent protein (GFP) fusion protein (Hof1p-GFP) at successive phases of the cell division cycle viewed by DIC (top left panel) or fluorescence (more...)

Is Hof1p a component of the contractile actomyosin ring? Or does Hof1p form a distinct ring? Time lapse imaging of cells expressing a Hof1p-GFP fusion protein revealed that the single Hof1p ring undergoes constriction coincident with actomyosin ring constriction (Fig. 3). However, while the contractile actomyosin ring constricts down to a dot and disappears, the Hof1p ring does not fully constrict. Hence, although the single Hof1p ring is in close apposition to the contractile actomyosin ring, they are two distinct rings.24,26-28,33,34

Figure 3. The Hof1p ring at the bud neck constricts, but not completely, during cytokinesis.

Figure 3

The Hof1p ring at the bud neck constricts, but not completely, during cytokinesis. Shown are cells as in Figure 2 viewed by DIC (left panels) or fluorescence optics (right panels) to visualize constriction (3-9) and disassembly (18) of the Hof1p-GFP single (more...)

How is Hof1p targeted to the bud neck? The observation that in M phase Hof1p localizes first to a large ring on the mother cell side and later to a similar ring on the daughter cell side of the bud neck suggested the possible involvement of septins in Hof1p targeting. Septins are essential for targeting of Hof1p to the bud neck because in a septin mutant (cdc12-6) Hof1p dissociates from the bud neck. In contrast, Hof1p localization does not require the actomyosin ring because mutant cells deficient in Myo1p fail to recruit F-actin to form an actomyosin ring yet retain the ability to form a Hof1p ring. All the information essential for directing Hof1p to the bud neck resides in the F-BAR domain as the PEST motif and SH3 are dispensable for Hof1p localization.24,26,27,35

Hof1p relocalization from two rings to a single ring at the bud neck is controlled, at least in part, by the Mitotic Exit Network (MEN), a set of protein kinases and phosphatases that coordinate successful chromosome segregation with exit from M-phase. MEN components include the protein kinases Cdc15p (not to be confused with the S. pombe PCH protein Cdc15), Dbf2p and Dbf20p (which have redundant functions), the protein phosphatase Cdc14p and the small regulatory GTPase Tem1p. cdc14 mutants arrest with two Hof1p rings, dbf2 dbf20 double mutants arrest in the process of converting from two rings to a single ring with an "hourglass-shaped" ring and cdc15 mutants arrest with half the cells displaying two rings and half displaying an "hour glass-shaped" ring (Fig. 3). Hence, cdc14 blocks earlier than dbf2 dbf20 and cdc15 blocks between the other two.27,37,38

Regulation of Hof1p by Phosphorylation

Hof1p is hyperphosphorylated late in the cell cycle, but not in cells arrested in exit from mitosis by cdc14, dbf2 dbf20, or cdc15. This suggests that either hyperphosphorylation occurs after exit from mitosis and Hof1p relocalization to a single ring or that these mutants have defects in Hof1p hyperphosphorylation. An important clue to the role of Hof1p hyperphosphorylation came from analysis of Hof1p degradation in cdc15 and other mutants defective in mitotic exit. In cdc15 mutant cells Hof1p degradation was strongly delayed, suggesting that hyperphosphorylation of Hof1p regulates its proteolytic degradation. PEST motifs are rich in serine and threonine residues that are potential sites of phosphorylation and in other proteins PEST motifs are known to be hyperphosphorylated. The PEST motif is likely the site of hyperphosphorylation in Hof1p as loss of the PEST motif prevents hyperphosphorylation.27,28,38

Phenotype of Hof1p-Deficient Cells

Perhaps unexpectedly, S. cerevisiae cells in which the HOF1 gene has been deleted (hof1Δ) are viable. This is in contrast to the situation in S. pombe where CDC15 is an essential gene. S. cerevisiae cells lacking Hof1p are, however, less robust than wild type cells and grow slowly or become inviable at higher temperatures (e.g., 37°C) depending on the genetic background. The Hof1p F-BAR domain is essential for growth at 37°C, however, the PEST motif and SH3 domain are dispensable.24-28,31,35,39

In Hof1p-deficient cells loss of viability is associated with formation of clumps and chains of three or more cells (Fig. 4). This suggests a defect in either cytokinesis (septum formation) or subsequent cell separation (septum maturation and cleavage). Enzymatic digestion of the cell wall does not convert clumps of hof1Δ cells to separate cells. When hof1Δ cells arrested at 37°C are examined, the absence of septum and a continuity of the cytoplasm between neighbouring cells in each chain or clump is apparent. The bud neck is 2-3-fold wider in hof1Δ relative to wild type cells, as reported for other cytokinesis mutants. Hence, the defect is in cytokinesis. The size of the cells continues to increase after arrest and some cells become multinucleate, which suggests a cell cycle defect rather than a biosynthetic defect.25-28

Figure 4. Cells lacking Hof1p exhibit severe defects in cytokinesis.

Figure 4

Cells lacking Hof1p exhibit severe defects in cytokinesis. Morphology of wild-type (left) and hof1Δ (right) cells grown in liquid culture. Note that wild-type cells form buds that always separate from the mother cells following cytokinesis while (more...)

In S. pombe Cdc15 has been reported to be essential for repolarization of cortical actin patches to the site of cell division after exit from M-phase.40 Cell polarity is not lost in Hof1p-deficient cells. Indeed, buds of Hof1p-deficient cells are often hyper-polarized (Fig. 4). However, cell wall chitin becomes mislocalized over the entire cell surface upon shif to 37°C rather than remaining concentrated in the bud neck as observed in wild type cells (Fig. 4). This suggests a defect in the ability of cell wall biosynthetic machinery to sense cell polarity.25-27 Two studies reported normal actin patch polarization in hof1Δ cells (even at 37°C) (Fig. 4).26,27 One study found that cortical actin patches polarize normally to small growing buds early in the cell cycle, but not to the bud neck during cytokinesis.25 Two other studies reported a severe loss of polarization of both cortical actin patches and cytoplasmic actin cables in hof1Δ cells.41,42 Loss of Hof1p is not reported to have major effects on the microtubule cytoskeleton.

Another possible explanation for the cytokinesis defects in Hof1p-deficient cells could be inefficient formation or dysfunction of the actomyosin ring. Myo1p localizes to a ring at the bud neck in Hof1p-deficient cells and this ring can still recruit F-actin.26,27 In one study the actomyosin ring tagged with Myo1p-GFP was reported to constrict more rapidly in Hof1p-deficient cells. In Hof1p-deficient cells the Myo1p-GFP ring appeared to move inwards from only one side of the bud neck and its fluorescence intensity appeared to decrease from this side during constriction. This is in contrast to wild type cells in which constriction occurs from both sides and culminates in the formation of a bright Myo1p-GFP dot in the middle of the bud neck.26 However, another study found no evidence for altered constriction of actomyosin rings in Hof1p-deficient cells.27 Interestingly, before the actomyosin ring forms (e.g., in cells blocked in exit from M) a wide F-actin belt is sometimes apparent on the mother cell side of the bud neck. This actin belt is of unknown function but may be a precursor of the actomyosin ring. This F-actin belt is absent in hof1Δ cells.34 In hof1Δ cells loss of the actin belt may be insufficient to block formation of the actomyosin ring in M but may afect its stability or function.

The importance of septin rings for cytokinesis ofered a possible explanation for the cytoki-nesis defects in hof1Δ cells. Perhaps Hof1p is essential for the assembly of septin rings. However, this is not the case. Septins still form a ring at the new bud site in G1 and then split into two rings in M phase in Hof1p-deficient cells. Furthermore, septin rings form in G1 before Hof1p is observed.26,27 In wild type cells the septin rings inherited by the mother cell and daughter cell at cytokinesis rapidly disappear. One study found that septin rings persist in hof1Δ cells at multiple sites of failed cytokinesis within a chain of cells. It was also found that over-expression of Hof1p dissociates septin rings.26 A later study, however, did not observe defects in septin ring formation, localization, or disassembly in hof1Δ cells.27

HOF1 Over-Expression Phenotype

Over-expression of HOF1 is lethal and the Hof1p over-expressing cells arrest as chains and clumps of connected cells, often with hyperpolarized buds.26,28 In cells subjected to prolonged HOF1 over-expression F-actin aggregates accumulate at the bud neck before M but actomyosin rings of normal appearance are only observed after entry into M. Hence, unlike CDC15 in S. pombe, over-expression of HOF1 in S. cerevisiae is insufficient to drive actomyosin ring formation in G2.26 Interestingly, over-expression of HOF1Δ PEST inhibits actomyosin ring constriction and delays disassembly.28

A Hof1p-Dependent Pathway of Cytokinesis Independent of the Cytokinetic Actomyosin Ring

The actomyosin contractile ring is dispensable for cytokinesis in S. cerevisiae, in contrast to S. pombe. This may be because S. cerevisiae, unlike S. pombe, has a constricted neck region due to its growth by budding, which may make cytokinesis easier to complete.22 Clearly, an actomyosin-ring independent pathway of cytokinesis exists in S. cerevisiae. The lack of apparent defect in actomyosin ring constriction, coupled with the fact that S. cerevisiae appears to have a second pathway of cytokinesis that is independent of the cytokinetic actomyosin ring prompted an investigation of whether Hof1p has a role in the actomyosin-ring independent pathway. Strikingly, while loss of Hof1p or the contractile actomyosin ring component Myo1p individually did not result in loss of viability, loss of both is lethal. Hence, Hof1p functions in the actomyosin-ring independent pathway of cytokinesis.25,27,34

What causes this delay in cytokinesis and cell separation in hof1Δ cells? The answer to this question is elusive. However, Vallen and coworkers27 have proposed a plausible model in which Hof1p acts as an adaptor to localize the enzymes that synthesize the septum so the septum forms at the proper time and place. In support of this possibility, Hof1p colocalizes with one chitin synthase in a large ring on the mother cell side of the bud neck in early M phase and with another chitin synthase in a small ring precisely at the bud neck after exit from mitosis. The observed asymmetric initiation of the septum and its poorly directed growth across the bud neck in hof1Δ cells are consistent with improper localization of chitin synthases.1,23,27 The molecular mechanisms by which Hof1p localizes chitin synthases at the bud neck are still unclear but likely involve a predicted integral membrane protein and cell polarity factor known as Skg6p. Skg6p acts antagonistically with Hof1p in cytokinesis.36

A Novel Function for the Hof1p SH3 Domain

What is the role of the Hof1p SH3 domain? The answer to this question came from studies of the actin patch protein Vrp1p. Vrp1p predominantly localizes to cortical actin patches.10,11 Despite this non-bud-neck localization, Vrp1p functions in the Hof1p-dependent pathway of cytokinesis. Loss of Vrp1p severely delays cytokinesis at 24°C and results in arrest of the majority of cells in cytokinesis at 37°C. In vrp1Δ cells arrested at 37°C Hof1p is delocalized from the bud neck in the majority of cells. As is the case with hof1Δ , vrp1Δ is synthetic lethal with mutations affecting the actomyosin ring, e.g., deletion of the gene encoding Myo1p (myo1Δ ). However, vrp1Δ also affects assembly and constriction of the actomyosin ring and is synthetic lethal with hof1Δ . Hence, Vrp1p functions in both actomyosin ring and Hof1p-dependent pathways of cytokinesis.31,32,34,35

An N-terminal Vrp1p fragment (N-Vrp1p, comprising residues 1-364) physically interacts with the Hof1p SH3 domain. N-Vrp1p lacks C-terminal sequences required for localization to cortical actin patches. Yet expression of this fragment (from the VRP1 promoter on a plasmid maintained at 2-5 copies per cell) is sufficient to restore efficient Hof1p localization to the bud neck and rescue cytokinesis at 37°C.31,32 Interaction with the Hof1p SH3 domain requires a proline-rich domain of Vrp1p known as the Hof One Trap (HOT) domain. The HOT domain comprises three proline-rich motifs (PRMs) that match the consensus PXPXXPSS (where P is proline, X is any amino acid and S is serine). Each of the three HOT domain PRMs is essential for strong interaction with the Hof1p SH3 domain.35 In the Hof1p SH3 domain Tyr608 is critical for interaction with Vrp1p.35 These interactions may be specifc to S. cerevisiae, as the S. pombe Cdc15 SH3 domain does not interact with the S. pombe WIP/ Vrp1p ortholog Vrp1.43

In cells lacking Vrp1p or with a mutated form of N-Vrp1p lacking a functional HOT domain Hof1p still localizes to rings at the bud neck at 24°C but it dissociates from the bud neck at 37°C resulting in arrest in cytokinesis. However, deletion of the Hof1p SH3 domain bypasses the requirement for the Vrp1p HOT domain for Hof1p localization to rings at the bud neck and cytokinesis at 37°C 35. Hence, rather than functioning as an adaptor to recruit Vrp1p to the bud neck, the Hof1p SH3 domain seems to confer Vrp1p-dependence on Hof1p localization to the bud neck. This explains the observation that although Hof1p localization to the bud neck does not require the SH3 domain, when the SH3 domain is present efficient Hof1p localization requires that its SH3 domain interact with Vrp1p. This suggests a regulatory switch linking Hof1p recruitment to the bud neck to other cellular events rather than a mechanical role in recruitment per se. What upstream signal activates this regulatory switch is not known.

Hof1p also directly interacts via its SH3 domain with both the formin-family proteins Bni1p and Bnr1p. The Formin Homology 1 (FH1) domain of Bnr1p is sufficient for this interaction, however the FH1 domain of Bni1p requires additional flanking sequences for interaction. Full-length Hof1p interacts with full-length Bnr1p only in the presence of GTP-bound Rho4p.25 The functional consequences of Hof1p-Bnr1p interaction are not known.

Bzz1p/Lsb7p

S. cerevisiae Bzz1p or Lsb7p is a 633 amino acid protein that contains, in addition to a more recently identifed N-terminal F-BAR domain, two C-terminal SH3 domains (Fig. 1). Bzz1p was identifed in a yeast two-hybrid screen with the yeast WASP-related protein Las17p that also picked up the yeast WIP-related protein Vrp1p, the endophilin- and amphiphysin-related protein Rvs167p and a number of then unknown proteins called Lsb1p to Lsb7p (for Las Seventeen Binding proteins). Bzz1p was also identifed by affinity purifcation of Las17p-associated proteins.44,45 Blast searches reveal Bzz1p orthologs in many other yeast species. Bzz1p homologs have also been identifed in fies, mice and humans where the protein is called nervous wreck (Nwk) after the Drosophila phenotype of a paralytic mutant exhibiting excessive growth of neuromuscular junction boutons.46,47 Bzz1p is thus one of a number of Las17p (WASP/WAVE family) ligands that could contribute to Las17p inhibition or activation in actin filament assembly and interact with proline—rich peptide stretches in other proteins such as Vrp1p and the type I unconventional myosins, Myo3p and Myo5p. These proteins comprise a macromolecular complex dependent on the Rho-family GTPase, Cdc42p.13,15,44,48-51

Bzz1p Subcellular Localization

Bzz1p is present throughout the cell cycle. Bzz1p, and in particular its SH3 domains, was shown by two-hybrid mapping to bind to multiple sites within the central proline—rich region of Las17p (aa∼ 300-500) and also to a small N-terminal fragment (aa 91-209). Confirmation of direct physical interaction was obtained in vitro by producing the relevant recombinant proteins and polypeptides and by co-immunoprecipitation.45,50 Bzz1-GFP fusion proteins localize to motile polarized cortical patches and show colocalization with both actin and Las17p. This localization is independent of polymerized actin because it is resistant to the actin polymerization inhibitor latrunculin-A. In the absence of the polarity protein Vrp1p or the type I myosins, Bzz1-GFP still localizes to cortical patches, however these patches are no longer polarized to sites of growth. In contrast, Bzz1-GFP localization is strictly dependent on Las17p because in a las17Δ strain Bzz1-GFP has a diffuse distribution throughout the cytoplasm and is rapidly degraded.45 Perhaps interaction with Las17p inhibits the ability of the PEST motif to target Bzz1p for degradation. Since Bzz1p has been found to interact in two-hybrid with Grr1p, it may be a substrate for SCF-targeted degradation like Hof1p.28,30,50

Bzz1p-deficient and Over-Expressing Cells

Deletion of the BZZ1 gene alone in three different strain backgrounds did not affect growth under a variety of conditions and at different temperatures including respiration on glycerol, sensitivity to cations, sorbitol, caffeine, calcofluor white (cell wall inhibitor), latrunculin-A (actin inhibitor), benomyl (microtubule inhibitor), capacity of haploid cells to mate and form diploids or capacity of diploids to sporulate and form haploids). Loss of Bzz1p also did not alter the distribution of cortical actin patches or actin cables throughout the cell cycle45 (and our unpublished data). Nor did deletion of BZZ1 appear to aggravate temperature sensitivity, osmosensitivity or actin polarization defects of a las17Δ mutant strain. However, the triple deletion of BZZ1 with the type I myosins, bzzΔ myo3Δ myo5Δ was lethal in a strain background where the myo3Δ myo5Δ double mutant is viable. The bzz1Δ myo5Δ double mutant, but not either single mutant, was sensitive to salt. The actin cytoskeleton becomes depolarized in response to salt stress in S. cerevisiae. In wild-type cells the actin cytoskeleton gradually recovers, but in the bzz1Δ myo5Δ double mutant it did not repolarize, a first indication that Bzz1p acts with Myo5p in actin function. The overexpression of BZZ1 inhibits growth at 37°C but no specifc actin cytoskeleton or cell cycle defects were observed.45

A Function for the Bzz1p SH3 Domain

The most direct evidence for Bzz1p function came from the demonstration in an in vitro polymerization assay that recombinant GST-Bzz1p, through its SH3 domains, could recruit and trigger a functional actin polymerization machinery like Las17p45 or the Myo5p C-terminal tail.52 This polymerization is dependent on functional Arp2p and Las17p, Vrp1p and the type I myosins. It was later shown that Bzz1p functions with Myo5p at the internalization stage of endocytosis. The double mutant has a mild defect in both fuid-phase and receptor-mediated endocytosis while vacuolar delivery of cargoes from both the endocytic and biosynthetic pathways appeared unaffected.53

Bzz1p Pathways

A number of studies in different laboratories have shown changing cortical actin patch composition and patch movement during Arp2/3-dependent actin polymerization and endocytosis. This paved the way for live imaging studies that established an order and timing of the molecular events and mechanisms of actin function during endocytosis.16,19,54-61 Interplay of four protein groups or modules is proposed. A coat protein group containing clathrin, Pan1p (a weak Arp2/3 activator), End3p and adaptors Sla1p and Sla2p first assembles at the plasma membrane, independently of actin. This complex later requires actin polymerization to disassemble. The actin protein group consists of the Arp2/3 complex, actin, the Arp2/3 activator Abp1p and other structural components needed to assemble branched actin meshworks. An activator protein group that remains at the membrane consists of Las17p and Vrp1p that recruit actin monomers and bind directly to Myo5p and the SH3-domain protein Bbc1p. Sla1p apparently inactivates Las17p until Bzz1p (and perhaps other proteins) arrive. The activator protein group also assembles into patches at the site of polymerization independently of actin but, like the coat protein group, requires actin polymerization to disassemble. Both the nucleation and motor activities of Myo5p are necessary for internalization. Finally, an amphiphysin protein group that contains the BAR domain proteins Rvs161p and Rvs167p arrives at the plasma membrane and almost immediately moves ∼ 100 nm inwards and dissipates, probably subsequent to vesicle fission.

Elegant confirmation of Bzz1p localization and its function in actin polymerization at these sites of endocytic internalization came from the studies by Sun et al. In vitro, purifed Bzz1p relieves Sla1p inhibition of Las17p and in vivo Bzz1-GFP is recruited to cortical patches just before actin is polymerized, consistent with a function in relieving prior Las17p inhibition by Sla1p (Fig. 5). However, initiation of actin polymerization is normal in a strain lacking Bzz1p; thus this activation by Bzz1p represents a redundant function.45,61 What upstream signals initiate Bzz1p recruitment to patches is not yet known.

Figure 5. The role of Bzz1p in a model for how actin assembly is regulated and produces force during endocytic internalization.

Figure 5

The role of Bzz1p in a model for how actin assembly is regulated and produces force during endocytic internalization. A) Examples of the fluorescence intensity of proteins at individual cortical sites were recorded from cells in which two cytoskeletal (more...)

SH3-containing PCH proteins may act at membranes other than the plasma membrane. A Bzz1p mammalian homolog, syndapin II, promotes vesicle budding at the Golgi membrane.62 As is the case for S. cerevisiae Hof1p, several possible mammalian homologs of Bzz1p such as syndapin, CIP4a, FBC17 and Toca-1 interact with formins.25,63 The FH1 domain of Bnr1p interacts with a polypeptide containing both SH3 domains of Bzz1p (but not the individual SH3 domains) in two-hybrid assays.50 However, the function of Bzz1p interaction with formins has not been explored. Finally, it would also seem that modification by phosphorylation is an emerging question as Bzz1p was identifed as a possible substrate for at least three different kinases,64 possibly implicating not only endocytic trafficking but also septin assembly, the protein kinase C pathway and the unfolded protein response.

The Rgd Proteins

Rgd1p and Rgd2p (for Related GAP domain) can be considered as PCH family proteins in an expanded definition, that is, all those containing an FCH or F-BAR domain. The term PCH was originally reserved for adaptor proteins containing both FCH and SH3 domains. Among ten S. cerevisiae RhoGAPs (GTPase Activating Protein of the small Rho GTPases), only Rgd1p and Rgd2p also have an F-BAR domain. Like in other PCH proteins, the FCH domain of Rgd1p and Rgd2p is located in the N-terminus and a GAP domain in the C-terminus. Rgd2p, but not Rgd1p, has a PEST motif in the C-terminal GAP domain (Fig. 1). The domain architecture of the Rgd proteins resembles that of the mammalian Rho GTPase subfamily containing p115 and srGAP (Slit-Robo GAP) proteins, with the intriguing diference that, in addition to the FCH and GAP domains, the mammalian proteins have a C-terminal SH3 domain. Sequence homology comparisons of 73 GAP domains places Rgd1p and Rgd2p on different branches of a GAP domain phylogenetic tree, consistent with functional data.65

Rgd1p

Rgd1p, discovered as a GAP domain protein by sequencing, interacts with Rho2p, Rho3p and Rho4p in a two-hybrid assay. It was shown to stimulate in vitro the GTPase activity of only Rho3p and Rho4p.66 Rgd1p was shown to functionally interact with Vrp1p and Las17p which receive signals from Cdc42p that regulates polarization of cell growth, including polarization of the actin cytoskeleton but also delivery of secretory vesicles to the plasma membrane.13 Arguments in favour of Rho3p and Rho4p being the targets of Rgd1p came from studies using inactive or constitutively active forms of these GTPases which provided evidence of their involvement in rgd1Δ vrp1Δ and rgd1Δ las17Δ synthetic lethality.14,67

Rgd1-GFP localized to patch-like structures at the base of the emerging bud and at the cell periphery that did not overlap significantly with actin patches.67 Work on the GTPase Rho3p suggests that it regulates actin polarity and directed secretion of exocytic vesicles from the mother cell to the bud specifically during polarized growth of the emerging bud. These functions are distinct from the docking and fusion of vesicles with the plasma membrane mediated by the exocyst, a multiple subunit complex that targets secretory vesicles to specific dockingsites on the plasma membrane. Genetic and cell biological approaches support a model in which both Rho3p and Cdc42p GTPases act via local activation of the exocyst.4,68 Rgd1p localization is consistent with the fact that Cdc42p and Rho3p are spatial regulators of exocytosis and have partially overlapping functions. Thus, Rgd1p appears to negatively regulate directed secretion and membrane fusion at the plasma membrane.

The protein kinase C (PKC) mitogen-activated protein (MAP) kinase pathway functions in cell wall remodelling during growth. Through a combination of genetic studies of synthetic lethality and suppression it was also shown that Rgd1p interacts with Mid2p and Wsc1p, transmembrane proteins that sense damage to the cell wall (i.e., act as sensors) upstream of the PKC pathway.69 Work in the same group proposed that the constitutive activation of Rho3p and Rho4p due to inactivation of Rgd1p at the same time as the cell wall sensor Wsc1p cause synthetic lethality, since inactivation of Rho3p and Rho4p rescued the synthetic lethality of the double mutant.70

Since the rgd1Δ deletion phenotype could be suppressed by Rho1p, Rho2p and genes in the PKC pathway, it has been proposed that Rgd1p decreases signaling via the PKC pathway.69,71 Transcriptional regulation of the RGD1 gene under different stress conditions showed activation of transcription at low pH and after heat and oxidative shocks. The general stress-activated transcription factors Msn2p and Msn4p, as well as Hog1p and Pbs2p from the osmosensory signaling HOG pathway acted on the basal RGD1 transcriptional level in normal and stress conditions.72 Rgd1p may be involved in yet another signalling pathway. A recent genome-wide study found that an rgd1Δ mutant shows increased sensitivity to endoplasmic reticulum (ER) stress.73 Three pathways, the unfolded protein response (UPR mediates cell survival during hypoxia and is required for tumor growth), the SLT2 mitogen-activated protein kinase (MAPK) pathway and the osmosensing MAPK pathway were required for survival during ER stress.73 Rgd1p is thus involved in several signal transduction pathways, principally through its negative regulation of the Rho3p and Rho4p GTPases. To date, Rgd1p is the only known negative regulator of Rho3p and Rho4p.

Rgd2p

Less is known about Rgd2p. Its domain structure is different from Rgd1p in that it has a DEP (Disheveled/Egl-10/Pleckstrin) domain between the FCH and GAP domains (Fig. 1). Roumanie et al. first showed evidence for a sixth functional Rho protein in yeast, the Cdc42/Rac-like GTPase Rho5p and demonstrated that Rgd2p has GAP activity on both Cdc42p and Rho5p.74 Lack of Rho5p gives rise to an increased activity of the PKC-dependent signal transduction pathway since a rho5Δ deletion strain shows increased resistance to drugs known to affect this pathway. Actin depolarization and repolarization after heat treatment in rho5Δ strains as well as in strains over-expressing an activated rho5 allele are further evidence for an effect on this pathway. The authors conclude that Rho5p acts as a negative regulator in the MAP kinase cascade, which differentiates between MAP kinase-dependent and -independent functions of Pkc1p.75 Rgd2p might crosstalk with Rgd1p through both Cdc42p and PKC pathways.

Perspectives

F-BAR domains in higher eukaryotic PCH proteins have been shown to mediate oligomer-ization and interaction with membrane phospholipids.76,77 By now the reader might well ask what is known about the F-BAR domains of the S. cerevisiae PCH proteins? The easy answer is almost nothing. How important is dimerization (as suggested by the coiled-coil domains) for yeast PCH protein function? Do these domains interact directly with membrane lipids to sense membrane curvature or participate in the induction of membrane deformation? With which lipids or phospholipids do the F-BAR domains interact? Is distinct lipid binding specificity responsible for the observed distinct subcellular localizations of Hof1p and Bzz1p? Is the F-BAR domain sufficient or are other domains required for membrane interaction, i.e., is there coincident detection of multiple signals as observed in BAR domain proteins? Preliminary data in our laboratories suggest that the Bzz1p F-BAR domain binds phosphorylated phospholipids but the Hof1p F-BAR domain does not (G. Ren, C. Orange, A. Munn and B. Winsor, unpublished data). However, to our knowledge the specificity of binding or functional capacity for tubulation of membrane liposomes in vitro has not been explored for any of the S. cerevisiae F-BAR domains or proteins. Bzz1p has been reported to interact with Bsp1p,50 a protein that acts as an adaptor to recruit the phosphoinositide phosphatases (synaptojanins) Inp52p and Inp53p to the cortical actin cytoskeleton. This might influence the phosphorylation of phosphoinositide pools and phosphoinositide-dependent recruitment of endocytic coat complexes to the plasma membrane.

The 3D crystal structure of several PCH proteins of higher eukaryotes have very recently been elucidated.78-80 3D structures are not yet available for any S. cerevisiae F-BAR domain or full-length PCH/F-BAR protein although Bzz1p SH3 domain structures have recently been submitted to PDB data bank (1zuu and 2A28) from M. Wilmanns laboratory (EMBL, Hamburg, Germany). It will be interesting to see how closely the yeast F-BAR domains resemble each other and the F-BAR domains of the higher eukaryotic PCH proteins. In particular, it will be exciting to learn whether they form banana-shaped dimers and whether they also exhibit a concentration of basic amino acid residues on one face.

There are also many exciting questions about the function of Hof1p in cytokinesis. How does the F-BAR domain promote cytokinesis—is it only via efects on chitin synthases and septum deposition? If so, why do PCH proteins in higher eukaryotes that lack cell walls and septa also promote cytokinesis? Why does the Hof1p SH3 domain act antagonistically to the F-BAR domain, i.e., inhibit localization of the F-BAR domain to the bud neck and cytokinesis—processes which do not require the SH3 domain? Is there an intrinsic or extrinsic cue that triggers the SH3 domain of Hof1p to dis-engage from the Vrp1p HOT domain and inhibit cytokinesis? Is this part of a cell cycle checkpoint? If so, what is the nature of this cue? Why would a cell arrest cytokinesis afer mitosis is already completed? Could a checkpoint monitor the proper segregation of a cytoplasmic organelle into the daughter cell/bud (e.g., a mitochondrion, ER, Golgi)? Is interaction of the Hof1p SH3 domain with the Vrp1p HOT domain regulated by phosphorylation of the critical flanking serine residues? What are the targets of the Hof1p SH3 domain whose inhibition results in a block of both pathways of cytokinesis? Is this regulatory mechanism specific to yeast or also present in higher eukaryotes? These are important questions and the answers are sure to reveal some surprises.

Acknowledgements

The authors are grateful to Erfei Bi and David Drubin's laboratory for permission to reproduce figures from their original articles and to colleagues for helpful suggestions. The authors were funded by NHMRC Project Grant 252750 and the Queensland State Government (to ALM) and by the EU Research Training Network (Contract 0036076) and Centre National de Recherche Scientifique and Universite Louis Pasteur core funding (to BW).

References

1.
Pringle JR, Broach BJ, Jones EW ed(s) 1997. Molecular and cellular biology of the yeast Saccharomyces: Life Cycle and Inheritance. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.
2.
Drubin DG, Nelson WJ. Origins of cell polarity. Cell. 1996;84(3):335–344. [PubMed: 8608587]
3.
Pruyne D, Bretscher A. Polarization of cell growth in yeast. I. Establishment and maintenance of polarity states. J Cell Sci. 2000;113(Pt 3):365–375. [PubMed: 10639324]
4.
Adamo JE, Moskow JJ, Gladfelter AS. et al. Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud. J Cell Biol. 2001;155(4):581–592. [PMC free article: PMC2198861] [PubMed: 11706050]
5.
Zhang X, Bi E, Novick P. et al. Cdc42 interacts with the exocyst and regulates polarized secretion. J Biol Chem. 2001;276(50):46745–46750. [PubMed: 11595741]
6.
Gladfelter AS, Bose I, Zyla TR. et al. Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p. J Cell Biol. 2002;156(2):315–326. [PMC free article: PMC2199227] [PubMed: 11807094]
7.
Madden K, Snyder M. Cell polarity and morphogenesis in budding yeast. Annu Rev Microbiol. 1998;52:687–744. [PubMed: 9891811]
8.
Tolliday N, Bouquin N, Li R. Assembly and regulation of the cytokinetic apparatus in budding yeast. Curr Opin Microbiol. 2001;4(6):690–695. [PubMed: 11731321]
9.
Longtine MS, Bi E. Regulation of septin organization and function in yeast. Trends Cell Biol. 2003;13(8):403–409. [PubMed: 12888292]
10.
Munn AL. Molecular requirements for the internalisation step of endocytosis: insights from yeast. Biochim Biophys Acta. 2001;1535(3):236–257. [PubMed: 11278164]
11.
Engqvist-Goldstein AE, Drubin DG. Actin assembly and endocytosis: from yeast to mammals. Annu Rev Cell Dev Biol. 2003;19:287–332. [PubMed: 14570572]
12.
Moseley JB, Goode BL. The yeast actin cytoskeleton: from cellular function to biochemical mechanism. Microbiol Mol Biol Rev. 2006;70(3):605–645. [PMC free article: PMC1594590] [PubMed: 16959963]
13.
Lechler T, Jonsdottir GA, Klee SK. et al. A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast. J Cell Biol. 2001;155(2):261–270. [PMC free article: PMC2198833] [PubMed: 11604421]
14.
Roumanie O, Peypouquet MF, Thoraval D. et al. Functional interactions between the VRP1-LAS17 and RHO3-RHO4 genes involved in actin cytoskeleton organization in Saccharomyces cerevisiae. Curr Genet. 2002;40(5):317–325. [PubMed: 11935222]
15.
Goode BL, Rodal AA. Modular complexes that regulate actin assembly in budding yeast. Curr Opin Microbiol. 2001;4(6):703–712. [PubMed: 11731323]
16.
Rodal AA, Manning AL, Goode BL. et al. Negative regulation of yeast WASp by two SH3 domain-containing proteins. Curr Biol. 2003;13(12):1000–1008. [PubMed: 12814545]
17.
Winsor B, Schiebel E. Review: an overview of the Saccharomyces cerevisiae microtubule and microfilament cytoskeleton. Yeast. 1997;13(5):399–434. [PubMed: 9153752]
18.
Pruyne D, Bretcher A. Polarization of cell growth in yeast. II. The role of the cortical actin cytoskeleton. J Cell Sci. 2000;113(4):571–585. [PubMed: 10652251]
19.
Kaksonen M, Sun Y, Drubin DG. A pathway for association of receptors, adaptors and actin during endocytic internalization. Cell. 2003;115(4):475–487. [PubMed: 14622601]
20.
Evangelista M, Zigmond S, Boone C. Formins: signaling effectors for assembly and polarization of actin filaments. J Cell Sci. 2003;116(Pt 13):2603–2611. [PubMed: 12775772]
21.
Lippincott J, Li R. Sequential assembly of myosin II, an IQGAP-like protein and filamentous actin to a ring structure involved in budding yeast cytokinesis. J Cell Biol. 1998;140(2):355–366. [PMC free article: PMC2132585] [PubMed: 9442111]
22.
Bi E, Maddox P, Lew DJ. et al. Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis. J Cell Biol. 1998;142(5):1301–1312. [PMC free article: PMC2149343] [PubMed: 9732290]
23.
Cabib E, Roh DH, Schmidt M. et al. The yeast cell wall and septum as paradigms of cell growth and morphogenesis. J Biol Chem. 2001;276(23):19679–19682. [PubMed: 11309404]
24.
Lippincott J, Li R. Involvement of PCH family proteins in cytokinesis and actin distribution. Microsc Res Tech. 2000;49(2):168–172. [PubMed: 10816256]
25.
Kamei T, Tanaka K, Hihara T. et al. Interaction of Bnr1p with a novel Src homology 3 domain-containing Hof1p. Implication in cytokinesis in Saccharomyces cerevisiae. J Biol Chem. 1998;273(43):28341–28345. [PubMed: 9774458]
26.
Lippincott J, Li R. Dual function of Cyk2, a cdc15/PSTPIP family protein, in regulating actomyosin ring dynamics and septin distribution. J Cell Biol. 1998;143(7):1947–1960. [PMC free article: PMC2175218] [PubMed: 9864366]
27.
Vallen EA, Caviston J, Bi E. Roles of Hof1p, Bni1p, Bnr1p and myo1p in cytokinesis in Saccharomyces cerevisiae. Mol Biol Cell. 2000;11(2):593–611. [PMC free article: PMC14796] [PubMed: 10679017]
28.
Blondel M, Bach S, Bamps S. et al. Degradation of Hof1 by SCF(Grr1) is important for actomyosin contraction during cytokinesis in yeast. EMBO J. 2005;24:1440–1452. [PMC free article: PMC1142548] [PubMed: 15775961]
29.
Spellman PT, Sherlock G, Zhang MQ, Iyer VR. et al. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol Biol Cell. 1998;9(12):3273–3297. [PMC free article: PMC25624] [PubMed: 9843569]
30.
Ito T, Chiba T, Ozawa R. et al. A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci USA. 2001;98(8):4569–4574. [PMC free article: PMC31875] [PubMed: 11283351]
31.
Naqvi SN, Feng Q, Boulton VJ, Zahn R. et al. Vrp1p functions in both actomyosin ring-dependent and Hof1p-dependent pathways of cytokinesis. Traffic. 2001;2(3):189–201. [PubMed: 11260524]
32.
Thanabalu T, Munn AL. Functions of Vrp1p in cytokinesis and actin patches are distinct and neither requires a WH2/V domain. EMBO J. 2001;20(24):6979–6989. [PMC free article: PMC125783] [PubMed: 11742975]
33.
Song S, Lee KS. A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis. J Cell Biol. 2001;152(3):451–469. [PMC free article: PMC2195991] [PubMed: 11157974]
34.
Norden C, Liakopoulos D, Barral Y. Dissection of septin actin interactions using actin overexpression in Saccharomyces cerevisiae. Mol Microbiol. 2004;53(2):469–483. [PubMed: 15228528]
35.
Ren G, Wang J, Brinkworth R. et al. Verprolin cytokinesis function mediated by the Hof one trap domain. Traffic. 2005;6(7):575–593. [PubMed: 15941409]
36.
Gandhi M, Goode BL, Chan CS. Four novel suppressors of gic1 gic2 and their roles in cytokinesis and polarized cell growth in Saccharomyces cerevisiae. Genetics. 2006;174(2):665–678. [PMC free article: PMC1602092] [PubMed: 16816427]
37.
Bardin AJ, Amon A. MEN and SIN: whats the difference? Nat Rev Mol Cell Biol. 2001;2(11):815–826. [PubMed: 11715048]
38.
Corbett M, Xiong Y, Boyne JR. et al. IQGAP and mitotic exit network (MEN) proteins are required for cytokinesis and repolarization of the actin cytoskeleton in the budding yeast, Saccharomyces cerevisiae. Eur J Cell Biol. 2006;85(11):1201–1215. [PubMed: 17005296]
39.
Fankhauser C, Reymond A, Cerutti L. et al. The S. pombe cdc15 gene is a key element in the reorganization of F-actin at mitosis. Cell. 1995;82(3):435–444. [PubMed: 7634333]
40.
Balasubramanian MK, McCollum D, Chang L. et al. Isolation and characterization of new fission yeast cytokinesis mutants. Genetics. 1998;149(3):1265–1275. [PMC free article: PMC1460233] [PubMed: 9649519]
41.
Thevissen K, Ayscough KR, Aerts AM. et al. Miconazole induces changes in actin cytoskeleton prior to reactive oxygen species induction in yeast. J Biol Chem. 2007;282(30):21592–21597. [PubMed: 17553796]
42.
Bonangelino CJ, Chavez EM, Bonifacino JS. Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae. Mol Biol Cell. 2002;13(7):2486–2501. [PMC free article: PMC117329] [PubMed: 12134085]
43.
Carnahan RH, Gould KL. The PCH family protein, Cdc15p, recruits two F-actin nucleation pathways to coordinate cytokinetic actin ring formation in Schizosaccharomyces pombe. J Cell Biol. 2003;162(5):851–862. [PMC free article: PMC2172828] [PubMed: 12939254]
44.
Madania A, Dumoulin P, Grava S. et al. The Saccharomyces cerevisiae homologue of human Wiskott-Aldrich syndrome protein Las17p interacts with the Arp2/3 complex. Mol Biol Cell. 1999;10(10):3521–3538. [PMC free article: PMC25621] [PubMed: 10512884]
45.
Soulard A, Lechler T, Spiridonov V. et al. Saccharomyces cerevisiae Bzz1p is implicated with type I myosins in actin patch polarization and is able to recruit actin-polymerizing machinery in vitro. Mol Cell Biol. 2002;22(22):7889–7906. [PMC free article: PMC134730] [PubMed: 12391157]
46.
Coyle IP, Koh YH, Lee WC. et al. Nervous wreck, an SH3 adaptor protein that interacts with Wsp, regulates synaptic growth in Drosophila. Neuron. 2004;41(4):521–534. [PubMed: 14980202]
47.
Chitu V, Stanley ER. Pombe Cdc15 homology (PCH) proteins: coordinators of membrane-cytoskeletal interactions. Trends Cell Biol. 2007;17(3):145–156. [PubMed: 17296299]
48.
Naqvi SN, Zahn R, Mitchell DA. et al. The WASp homologue Las17p functions with the WIP homologue End5p/verprolin and is essential for endocytosis in yeast. Curr Biol. 1998;8(17):959–962. [PubMed: 9742397]
49.
Evangelista M, Klebl BM, Tong AH. et al. A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p and the Arp2/3 complex. J Cell Biol. 2000;148(2):353–362. [PMC free article: PMC2174279] [PubMed: 10648568]
50.
Tong AH, Drees B, Nardelli G. et al. A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules. Science. 2002;295(5553):321–324. [PubMed: 11743162]
51.
Gavin AC, Bosche M, Krause R. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature. 2002;415(6868):141–147. [PubMed: 11805826]
52.
Geli MI, Lombardi R, Schmelzl B. et al. An intact SH3 domain is required for myosin I-induced actin polymerization. Embo J. 2000;19(16):4281–4291. [PMC free article: PMC302045] [PubMed: 10944111]
53.
Soulard A, Friant S, Fitterer C. et al. The WASP/Las17p-interacting protein Bzz1p functions with Myo5p in an early stage of endocytosis. Protoplasma. 2005;226(1-2):89–101. [PubMed: 16231105]
54.
Huckaba TM, Gay AC, Pantalena LF. et al. Live cell imaging of the assembly, disassembly and actin cable-dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae. J Cell Biol. 2004;167(3):519–530. [PMC free article: PMC2172478] [PubMed: 15534003]
55.
Jonsdottir GA, Li R. Dynamics of yeast Myosin I: evidence for a possible role in scission of endocytic vesicles. Curr Biol. 2004;14(17):1604–1609. [PubMed: 15341750]
56.
Young ME, Cooper JA, Bridgman PC. Yeast actin patches are networks of branched actin filaments. J Cell Biol. 2004;166(5):629–635. [PMC free article: PMC2172413] [PubMed: 15337772]
57.
Rodal AA, Kozubowski L, Goode BL. et al. Actin and septin ultrastructures at the budding yeast cell cortex. Mol Biol Cell. 2005;16(1):372–384. [PMC free article: PMC539180] [PubMed: 15525671]
58.
Newpher TM, Smith RP, Lemmon V. et al. In vivo dynamics of clathrin and its adaptor-dependent recruitment to the actin-based endocytic machinery in yeast. Dev Cell. 2005;9(1):87–98. [PubMed: 15992543]
59.
Kaksonen M, Toret CP, Drubin DG. A modular design for the clathrin- and actin-mediated endocytosis machinery. Cell. 2005;123(2):305–320. [PubMed: 16239147]
60.
Kim K, Galletta BJ, Schmidt KO. et al. Actin-based motility during endocytosis in budding yeast. Mol Biol Cell. 2006;17(3):1354–1363. [PMC free article: PMC1382323] [PubMed: 16394096]
61.
Sun Y, Martin AC, Drubin DG. Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity. Dev Cell. 2006;11(1):33–46. [PubMed: 16824951]
62.
Kessels MM, Dong J, Leibig W. et al. Complexes of syndapin II with dynamin II promote vesicle formation at the trans-Golgi network. J Cell Sci. 2006;119(Pt 8):1504–1516. [PubMed: 16551695]
63.
Aspenstrom P, Richnau N, Johansson AS. The diaphanous-related formin DAAM1 collaborates with the Rho GTPases RhoA and Cdc42, CIP4 and Src in regulating cell morphogenesis and actin dynamics. Exp Cell Res. 2006;312(12):2180–2194. [PubMed: 16630611]
64.
Ptacek J, Devgan G, Michaud G. et al. Global analysis of protein phosphorylation in yeast. Nature. 2005;438(7068):679–684. [PubMed: 16319894]
65.
Tcherkezian J, Lamarche-Vane N. Current knowledge of the large RhoGAP family of proteins. Biol Cell. 2007;99(2):67–86. [PubMed: 17222083]
66.
Doignon F, Weinachter C, Roumanie O. et al. The yeast Rgd1p is a GTPase activating protein of the Rho3 and Rho4 proteins. FEBS Lett. 1999;459(3):458–462. [PubMed: 10526184]
67.
Roumanie O, Peypouquet MF, Bonneu M. et al. Evidence for the genetic interaction between the actin-binding protein Vrp1 and the RhoGAP Rgd1 mediated through Rho3p and Rho4p in Saccharomyces cerevisiae. Mol Microbiol. 2000;36(6):1403–1414. [PubMed: 10931290]
68.
Adamo JE, Rossi G, Brennwald P. The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity. Mol Biol Cell. 1999;10(12):4121–4133. [PMC free article: PMC25747] [PubMed: 10588647]
69.
de Bettignies G, Barthe C, Morel C. et al. RGD1 genetically interacts with MID2 and SLG1, encoding two putative sensors for cell integrity signalling in Saccharomyces cerevisiae. Yeast. 1999;15(16):1719–1731. [PubMed: 10590461]
70.
Fernandes H, Roumanie O, Claret S. et al. The Rho3 and Rho4 small GTPases interact functionally with Wsc1p, a cell surface sensor of the protein kinase C cell-integrity pathway in Saccharomyces cerevisiae. Microbiology. 2006;152(Pt 3):695–708. [PubMed: 16514150]
71.
de Bettignies G, Thoraval D, Morel C. et al. Overactivation of the protein kinase C-signaling pathway suppresses the defects of cells lacking the Rho3/Rho4-GAP Rgd1p in Saccharomyces cerevisiae. Genetics. 2001;159(4):1435–1448. [PMC free article: PMC1461911] [PubMed: 11779787]
72.
Gatti X, de Bettignies G, Claret S. et al. RGD1, encoding a RhoGAP involved in low-pH survival, is an Msn2p/Msn4p regulated gene in Saccharomyces cerevisiae. Gene. 2005;351:159–169. [PubMed: 15922872]
73.
Chen Y, Feldman DE, Deng C. et al. Identifcation of mitogen-activated protein kinase signaling pathways that confer resistance to endoplasmic reticulum stress in Saccharomyces cerevisiae. Mol Cancer Res. 2005;3(12):669–677. [PubMed: 16380504]
74.
Roumanie O, Weinachter C, Larrieu I. et al. Functional characterization of the Bag7, Lrg1 and Rgd2 RhoGAP proteins from Saccharomyces cerevisiae. FEBS Lett. 2001;506(2):149–156. [PubMed: 11591390]
75.
Schmitz HP, Huppert S, Lorberg A. et al. Rho5p downregulates the yeast cell integrity pathway. J Cell Sci. 2002;115(Pt 15):3139–3148. [PubMed: 12118069]
76.
Itoh T, Erdmann KS, Roux A. et al. Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev Cell. 2005;9(6):791–804. [PubMed: 16326391]
77.
Tsujita K, Suetsugu S, Sasaki N. et al. Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. J Cell Biol. 2006;172(2):269–279. [PMC free article: PMC2063556] [PubMed: 16418535]
78.
Henne WM, Kent HM, Ford MG. et al. Structure and analysis of FCHo2 F-BAR domain: a dimerizing and membrane recruitment module that effCects membrane curvature. Structure. 2007;15(7):839–852. [PubMed: 17540576]
79.
Frost A, De Camilli P, Unger VM. F-BAR proteins join the BAR family fold. Structure. 2007;15(7):751–753. [PubMed: 17637334]
80.
Shimada A, Niwa H, Tsujita K. et al. Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis. Cell. 2007;129(4):761–772. [PubMed: 17512409]
81.
Lupas A, Van Dyke M, Stock J. Predicting coiled coils from protein sequences. Science. 1991;252(5009):1162–1164. [PubMed: 2031185]
82.
Rogers S, Wells R, Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science. 1986;234(4774):364–368. [PubMed: 2876518]
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