Vesicle delivery of Cdc42 has been proposed as an important mechanism for generating and maintaining Cdc42 polarity at the plasma membrane. This mechanism requires the density of Cdc42 on secretory vesicles to be equal to or higher than the plasma membrane polarity cap. Using a novel method to estimate Cdc42 levels on post-Golgi secretory vesicles in intact yeast cells, we: 1) determined that endocytosis plays an important role in Cdc42’s association with secretory vesicles 2) found that a GFP-tag placed on the N-terminus of Cdc42 negatively impacts this vesicle association and 3) quantified the surface densities of Cdc42 on post-Golgi vesicles which revealed that the vesicle density of Cdc42 is three times more dilute than that at the polarity cap. This work suggests that the immediate consequence of secretory vesicle fusion with the plasma membrane polarity cap is to dilute the local Cdc42 surface density. This provides strong support for the model in which vesicle trafficking acts to negatively regulate Cdc42 polarity on the cell surface while also providing a means to recycle Cdc42 between the cell surface and internal membrane locations.
We here report the first comparative proteomics of purified yeast post-Golgi vesicles (PGVs). Vesicle samples isolated from PGV-accumulating sec6-4 mutants were treated with isobaric tags (iTRAQ) for subsequent quantitative tandem mass spectrometric analysis of protein content. After background subtraction, a total of 66 vesicle-associated proteins were identified, including known or assumed vesicle residents as well as a fraction not previously known to be PGV associated. Vesicles isolated from cells lacking the polarity protein Sro7p contained essentially the same catalogue of proteins but showed a reduced content of a subset of cargo proteins, in agreement with a previously shown selective role for Sro7p in cargo sorting.
exocytosis; Golgi; membrane trafficking; proteomics; vesicles; iTRAQ
The yeast type V myosin, Myo2, and the lethal giant larvae homologue, Sro7, are important players in polarized exocytosis. This paper article characterizes the role of Myo2 both in recruiting Sro7 to sites of polarized growth and in negatively regulating a Sec4-dependent vesicle-clustering activity of Sro7.
Lgl family members play an important role in the regulation of cell polarity in eukaryotic cells. The yeast homologues Sro7 and Sro77 are thought to act downstream of the Rab GTPase Sec4 to promote soluble N-ethylmaleimide–sensitive factor adaptor protein receptor (SNARE) function in post-Golgi transport. In this article, we characterize the interaction between Sro7 and the type V myosin Myo2 and show that this interaction is important for two distinct aspects of Sro7 function. First, we show that this interaction plays a positive role in promoting the polarized localization of Sro7 to sites of active growth. Second, we find evidence that Myo2 negatively regulates Sro7 function in vesicle clustering. Mutants in either Myo2 or Sro7 that are defective for this interaction show hypersensitivity to Sro7 overexpression, which results in Sec4-dependent accumulation of large groups of vesicles in the cytoplasm. This suggests that Myo2 serves a dual function, to both recruit Sro7 to secretory vesicles and inhibit its Rab-dependent tethering activity until vesicles reach the plasma membrane. Thus Sro7 appears to coordinate the spatial and temporal nature of both Rab-dependent tethering and SNARE-dependent membrane fusion of exocytic vesicles with the plasma membrane.
Rho family GTPases are critical regulators in determining and maintaining cell polarity. In Saccharomyces cerevisiae, Rho3 and Cdc42 play important but distinct roles in regulating polarized exocytosis and overall polarity. Cdc42 is highly polarized during bud emergence and is specifically required for exocytosis at this stage. In contrast, Rho3 appears to play an important role during the isotropic growth of larger buds. Using a novel monoclonal antibody against Rho3, we find that Rho3 localizes to the cell surface in a dispersed pattern which is clearly distinct from that of Cdc42. Using chimeric forms of these GTPases, we demonstrate that a small region at the N terminus is necessary and sufficient to confer Rho3 localization and function onto Cdc42. Analysis of this domain reveals two essential elements responsible for distinguishing function. First, palmitoylation of a cysteine residue by the Akr1 palmitoyltransferase is required both for the switch of function and the switch of localization properties of this domain. Second, two basic residues distal to the palmitoylation site are required for regulating binding affinity with the Exo70 and Sec3 effectors. This demonstrates the importance of localization and effector binding in determining how these GTPases evolved specific functions at distinct stages of polarized growth.
At steady state, most Rho GTPases are bound in the cytosol to Rho Guanine nucleotide Dissociation Inhibitors (RhoGDI) 1. RhoGDIs have generally been considered to passively hold Rho proteins in an inactive state within the cytoplasm. Here we describe an evolutionarily conserved mechanism by which RhoGDI1 controls the homeostasis of Rho proteins in eukaryotic cells. We found that depletion of RhoGDI1 promotes misfolding and degradation of the cytosolic geranylgeranylated pool of Rho GTPases while unexpectedly activating the remaining membrane-bound fraction. Since RhoGDI1 levels are limiting, and Rho proteins compete for binding to RhoGDI1, overexpression of an exogenous Rho GTPase displaces endogenous Rho proteins bound to RhoGDI1, inducing their degradation and inactivation. These results raise important questions about the conclusions drawn from studies that manipulate Rho protein levels. In many cases the response observed may arise not simply from the overexpression per se, but from additional effects on the levels and activity of other Rho GTPases due to competition for binding to RhoGDI1, and may require a re-evaluation of previously published studies that rely exclusively on these techniques.
Genetic and biochemical evidence is presented that the Exo70 subunit of the exocyst is a direct effector for both Rho3 and Cdc42 GTPases in yeast. Prenylation of these GTPases both promotes the interaction and affects the site of binding within Exo70. Thus, interaction of the Rho GTPases with Exo70 is a key event in spatial regulation of exocytosis.
The Rho3 and Cdc42 members of the Rho GTPase family are important regulators of exocytosis in yeast. However, the precise mechanism by which they regulate this process is controversial. Here, we present evidence that the Exo70 component of the exocyst complex is a direct effector of both Rho3 and Cdc42. We identify gain-of-function mutants in EXO70 that potently suppress mutants in RHO3 and CDC42 defective for exocytic function. We show that Exo70 has the biochemical properties expected of a direct effector for both Rho3 and Cdc42. Surprisingly, we find that C-terminal prenylation of these GTPases both promotes the interaction and influences the sites of binding within Exo70. Finally, we demonstrate that the phenotypes associated with novel loss-of-function mutants in EXO70, are entirely consistent with Exo70 as an effector for both Rho3 and Cdc42 function in secretion. These data suggest that interaction with the Exo70 component of the exocyst is a key event in spatial regulation of exocytosis by Rho GTPases.
Exocytosis is the major mechanism by which new membrane components are delivered to the cell surface. In most, if not all, eukaryotic cells this is also a highly spatially regulated process that is tightly coordinated with the overall polarity of a cell. The Rho/Cdc42 family of GTPases and the lethal giant larvae/Sro7 family are two highly conserved families of proteins which appear to have dual functions both in cell polarity and exocytosis. Analysis of their functions has begun to unravel the coordination between these processes and propose a model for polarized vesicle docking and fusion at the site of asymmetric cell growth.
Rho GTPases; Lgl; exocytosis; cell polarity
The SRO7/SOP1 encoded tumor suppressor homologue of Saccharomyces cerevisiae is required for maintenance of ion homeostasis in cells exposed to NaCl stress. Here we show that the NaCl sensitivity of the sro7Δ mutant is due to defective sorting of Ena1p, the main sodium pump in yeast. On exposure of sro7Δ mutants to NaCl stress, Ena1p fails to be targeted to the cell surface, but is instead routed to the vacuole for degradation via the multivesicular endosome pathway. SRO7-deficient mutants accumulate post-Golgi vesicles at high salinity, in agreement with a previously described role for Sro7p in late exocytosis. However, Ena1p is not sorted into these post-Golgi vesicles, in contrast to what is observed for the vesicles that accumulate when exocytosis is blocked in sec6-4 mutants at high salinity. These observations imply that Sro7p has a previously unrecognized role for sorting of specific proteins into the exocytic pathway. Screening for multicopy suppressors identified RSN1, encoding a transmembrane protein of unknown function. Overexpression of RSN1 restores NaCl tolerance of sro7Δ mutants by retargeting Ena1p to the plasma membrane. We propose a model in which blocked exocytic sorting in sro7Δ mutants, gives rise to quality control-mediated routing of Ena1p to the vacuole.
Rab guanosine triphosphatases regulate intracellular membrane traffic by binding specific effector proteins. The yeast Rab Sec4p plays multiple roles in the polarized transport of post-Golgi vesicles to, and their subsequent fusion with, the plasma membrane, suggesting the involvement of several effectors. Yet, only one Sec4p effector has been documented to date: the exocyst protein Sec15p. The exocyst is an octameric protein complex required for tethering secretory vesicles, which is a prerequisite for membrane fusion. In this study, we describe the identification of a second Sec4p effector, Sro7p, which is a member of the lethal giant larvae tumor suppressor family. Sec4-GTP binds to Sro7p in cell extracts as well as to purified Sro7p, and the two proteins can be coimmunoprecipitated. Furthermore, we demonstrate the formation of a ternary complex of Sec4-GTP, Sro7p, and the t-SNARE Sec9p. Genetic data support our conclusion that Sro7p functions downstream of Sec4p and further imply that Sro7p and the exocyst share partially overlapping functions, possibly in SNARE regulation.
Rho GTPases are important regulators of polarity in eukaryotic cells. In yeast they are involved in regulating the docking and fusion of secretory vesicles with the cell surface. Our analysis of a Rho3 mutant that is unable to interact with the Exo70 subunit of the exocyst reveals a normal polarization of the exocyst complex as well as other polarity markers. We also find that there is no redundancy between the Rho3–Exo70 and Rho1–Sec3 pathways in the localization of the exocyst. This suggests that Rho3 and Cdc42 act to polarize exocytosis by activating the exocytic machinery at the membrane without the need to first recruit it to sites of polarized growth. Consistent with this model, we find that the ability of Rho3 and Cdc42 to hydrolyze GTP is not required for their role in secretion. Moreover, our analysis of the Sec3 subunit of the exocyst suggests that polarization of the exocyst may be a consequence rather than a cause of polarized exocytosis.
The tumor suppressor lethal giant larvae (Lgl) plays a critical role in epithelial cell polarization. However, the molecular mechanism by which Lgl carries out its functions is unclear. In this study, we report that the yeast Lgl proteins Sro7p and Sro77p directly interact with Exo84p, which is a component of the exocyst complex that is essential for targeting vesicles to specific sites of the plasma membrane for exocytosis, and that this interaction is important for post-Golgi secretion. Genetic analyses demonstrate a molecular pathway from Rab and Rho GTPases through the exocyst and Lgl to SNAREs, which mediate membrane fusion. We also found that overexpression of Lgl and t-SNARE proteins not only improves exocytosis but also rescues polarity defects in exocyst mutants. We propose that, although Lgl is broadly distributed in the cells, its localized interaction with the exocyst and kinetic activation are important for the establishment and reenforcement of cell polarity.
Kin1 and Kin2 are Saccharomyces cerevisiae counterparts of Par-1, the Caenorhabditis elegans kinase essential for the establishment of polarity in the one cell embryo. Here, we present evidence for a novel link between Kin1, Kin2, and the secretory machinery of the budding yeast. We isolated KIN1 and KIN2 as suppressors of a mutant form of Rho3, a Rho-GTPase acting in polarized trafficking. Genetic analysis suggests that KIN1 and KIN2 act downstream of the Rab-GTPase Sec4, its exchange factor Sec2, and several components of the vesicle tethering complex, the Exocyst. We show that Kin1 and Kin2 physically interact with the t-SNARE Sec9 and the Lgl homologue Sro7, proteins acting at the final stage of exocytosis. Structural analysis of Kin2 reveals that its catalytic activity is essential for its function in the secretory pathway and implicates the conserved 42-amino acid tail at the carboxy terminal of the kinase in autoinhibition. Finally, we find that Kin1 and Kin2 induce phosphorylation of t-SNARE Sec9 in vivo and stimulate its release from the plasma membrane. In summary, we report the finding that yeast Par-1 counterparts are associated with and regulate the function of the exocytic apparatus via phosphorylation of Sec9.
Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell–cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position.
EMK1; MARK2; MDCK; WIFB; apical surface
The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.
Cdc42; Rho; GTPases; exocytosis; cell polarity
The Drosophila tumor suppressor protein lethal (2) giant
larvae [l(2)gl] is involved in the establishment of epithelial
cell polarity during development. Recently, a yeast homolog of the
protein has been shown to interact with components of the post-Golgi
exocytic machinery and to regulate a late step in protein secretion.
Herein, we characterize a mammalian homolog of l(2)gl, called Mlgl, in
the epithelial cell line Madin-Darby canine kidney (MDCK). Consistent
with a role in cell polarity, Mlgl redistributes from a cytoplasmic
localization to the lateral membrane after contact-naive MDCK cells
make cell-cell contacts and establish a polarized phenotype.
Phosphorylation within a highly conserved region of Mlgl is required to
restrict the protein to the lateral domain, because a recombinant
phospho-mutant is distributed in a nonpolar manner. Membrane-bound Mlgl
from MDCK cell lysates was coimmunoprecipitated with syntaxin 4, a
component of the exocytic machinery at the basolateral membrane, but
not with other plasma membrane soluble
N-ethylmaleimide-sensitive factor attachment
receptor (SNARE) proteins that are either absent from or not
restricted to the basolateral membrane domain. These data suggest that
Mlgl contributes to apico-basolateral polarity by regulating
Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.
cytoskeleton; Rho proteins; secretion; cell polarity; endocytosis
We have identified a pair of related yeast proteins, Sro7p and Sro77p, based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, lethal giant larvae and to the neuronal syntaxin–binding protein, tomosyn. SRO7 and SRO77 have redundant functions as loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. We show that similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion. This demonstrates that the primary function of Sro7/77, and likely all members of the lethal giant larvae family, is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.
exocytosis; SNARE complex; cell polarity; tumor suppressor; tomosyn
Budding yeast grow asymmetrically by the polarized delivery of proteins and lipids to specific sites on the plasma membrane. This requires the coordinated polarization of the actin cytoskeleton and the secretory apparatus. We identified Rho3 on the basis of its genetic interactions with several late-acting secretory genes. Mutational analysis of the Rho3 effector domain reveals three distinct functions in cell polarity: regulation of actin polarity, transport of exocytic vesicles from the mother cell to the bud, and docking and fusion of vesicles with the plasma membrane. We provide evidence that the vesicle delivery function of Rho3 is mediated by the unconventional myosin Myo2 and that the docking and fusion function is mediated by the exocyst component Exo70. These data suggest that Rho3 acts as a key regulator of cell polarity and exocytosis, coordinating several distinct events for delivery of proteins to specific sites on the cell surface.
The crystal structure of the synaptic SNARE complex reveals a
parallel four-helix coiled-coil arrangement; buried in the hydrophobic
core of the complex is an unusual ionic layer composed of three
glutamines and one arginine, each provided by a separate α-helix. The
presence of glutamine or arginine residues in this position is highly
conserved across the t- and v-SNARE families, and it was recently
suggested that a 3Q:1R ratio is likely to be a general feature
common to all SNARE complexes. In this study, we have used genetic and
biochemical assays to test this prediction with the yeast exocytic
SNARE complex. We have determined that the relative position of Qs and
Rs within the layer is not critical for biological activity and that
Q-to-R substitutions in the layer reduce complex stability and result
in lethal or conditional lethal growth defects. Surprisingly, SNARE
complexes composed of four glutamines are fully functional for assembly
in vitro and exocytic function in vivo. We conclude that the 3Q:1R
layer composition is not required within the yeast exocytic SNARE
complex because complexes containing four Q residues in the ionic layer
appear by all criteria to be functionally equivalent. The unexpected
flexibility of this layer suggests that there is no strict requirement
for the 3Q:1R combination and that the SNARE complexes at other stages
of transport may be composed entirely of Q-SNAREs or other noncanonical