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1.  Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature 
Molecular Biology of the Cell  2014;25(21):3389-3400.
In yeast, the oxysterol-binding proteins Osh1–Osh7 are collectively needed to maintain the normal distribution of PI4P. Osh4p is recruited to secretory vesicles in a PI4P-dependent manner and plays an important role in vesicle maturation.
Phosphatidylinositol-4-phosphate (PI4P) is produced on both the Golgi and the plasma membrane. Despite extensive vesicular traffic between these compartments, genetic analysis suggests that the two pools of PI4P do not efficiently mix with one another. Several lines of evidence indicate that the PI4P produced on the Golgi is normally incorporated into secretory vesicles, but the fate of that pool has been unclear. We show here that in yeast the oxysterol-binding proteins Osh1–Osh7 are collectively needed to maintain the normal distribution of PI4P and that Osh4p is critical in this function. Osh4p associates with secretory vesicles at least in part through its interaction with PI4P and is needed, together with lipid phosphatases, to reduce the level of PI4P as vesicles approach sites of exocytosis. This reduction in PI4P is necessary for a switch in the regulation of the Sec4p exchange protein, Sec2p, from an interaction with the upstream Rab, Ypt31/32, to an interaction with a downstream Sec4p effector, Sec15p. Spatial regulation of PI4P levels thereby plays an important role in vesicle maturation.
PMCID: PMC4214785  PMID: 25165144
2.  The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth 
Molecular Biology of the Cell  2014;25(8):1312-1326.
The Neurospora crassa exocyst presents two distinct localization patterns. EXO-70 and -84 colocalize with a region of the Spitzenkörper occupied by secretory macrovesicles. In contrast, SEC-3, -5, -6, -8, and -15 localize distinctively at the apical plasma membrane.
Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.
PMCID: PMC3982996  PMID: 24523289
3.  The synaptobrevin homologue Snc2p recruits the exocyst to secretory vesicles by binding to Sec6p 
The Journal of Cell Biology  2013;202(3):509-526.
The exocyst is recruited to secretory vesicles by the combinatorial signals of Sec4-GTP and the Snc proteins to confer both specificity and directionality to vesicular traffic.
A screen for mutations that affect the recruitment of the exocyst to secretory vesicles identified genes encoding clathrin and proteins that associate or colocalize with clathrin at sites of endocytosis. However, no significant colocalization of the exocyst with clathrin was seen, arguing against a direct role in exocyst recruitment. Rather, these components are needed to recycle the exocytic vesicle SNAREs Snc1p and Snc2p from the plasma membrane into new secretory vesicles where they act to recruit the exocyst. We observe a direct interaction between the exocyst subunit Sec6p and the latter half of the SNARE motif of Snc2p. An snc2 mutation that specifically disrupts this interaction led to exocyst mislocalization and a block in exocytosis in vivo without affecting liposome fusion in vitro. Overexpression of Sec4p partially suppressed the exocyst localization defects of mutations in clathrin and clathrin-associated components. We propose that the exocyst is recruited to secretory vesicles by the combinatorial signals of Sec4-GTP and the Snc proteins. This could help to confer both specificity and directionality to vesicular traffic.
PMCID: PMC3734085  PMID: 23897890
4.  Different polarisome components play distinct roles in Slt2p-regulated cortical ER inheritance in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2013;24(19):3145-3154.
Slt2p kinase activity controls cortical ER inheritance by regulating the association of the ER with the actin-based cytoskeleton. The polarisome complex affects ER inheritance through its effects on Slt2p, with different components playing distinct roles: some are required for Slt2p retention at the bud tip, whereas others affect Slt2p activation.
Ptc1p, a type 2C protein phosphatase, is required for a late step in cortical endoplasmic reticulum (cER) inheritance in Saccharomyces cerevisiae. In ptc1Δ cells, ER tubules migrate from the mother cell and contact the bud tip, yet fail to spread around the bud cortex. This defect results from the failure to inactivate a bud tip–associated pool of the cell wall integrity mitogen-activated protein kinase, Slt2p. Here we report that the polarisome complex affects cER inheritance through its effects on Slt2p, with different components playing distinct roles: Spa2p and Pea2p are required for Slt2p retention at the bud tip, whereas Bni1p, Bud6p, and Sph1p affect the level of Slt2p activation. Depolymerization of actin relieves the ptc1Δ cER inheritance defect, suggesting that in this mutant the ER becomes trapped on the cytoskeleton. Loss of Sec3p also blocks ER inheritance, and, as in ptc1Δ cells, this block is accompanied by activation of Slt2p and is reversed by depolymerization of actin. Our results point to a common mechanism for the regulation of ER inheritance in which Slt2p activity at the bud tip controls the association of the ER with the actin-based cytoskeleton.
PMCID: PMC3784387  PMID: 23924898
5.  Transposable element evolution in Heliconius suggests genome diversity within Lepidoptera 
Mobile DNA  2013;4:21.
Transposable elements (TEs) have the potential to impact genome structure, function and evolution in profound ways. In order to understand the contribution of transposable elements (TEs) to Heliconius melpomene, we queried the H. melpomene draft sequence to identify repetitive sequences.
We determined that TEs comprise ~25% of the genome. The predominant class of TEs (~12% of the genome) was the non-long terminal repeat (non-LTR) retrotransposons, including a novel SINE family. However, this was only slightly higher than content derived from DNA transposons, which are diverse, with several families having mobilized in the recent past. Compared to the only other well-studied lepidopteran genome, Bombyx mori, H. melpomene exhibits a higher DNA transposon content and a distinct repertoire of retrotransposons. We also found that H. melpomene exhibits a high rate of TE turnover with few older elements accumulating in the genome.
Our analysis represents the first complete, de novo characterization of TE content in a butterfly genome and suggests that, while TEs are able to invade and multiply, TEs have an overall deleterious effect and/or that maintaining a small genome is advantageous. Our results also hint that analysis of additional lepidopteran genomes will reveal substantial TE diversity within the group.
PMCID: PMC4016481  PMID: 24088337
Heliconius melpomene; Lepidopteran; Butterfly; Transposable elements; Genomic deletions
6.  Role of Rab GTPases in Membrane Traffic and Cell Physiology 
Physiological reviews  2011;91(1):119-149.
Intracellular membrane traffic defines a complex network of pathways that connects many of the membrane-bound organelles of eukaryotic cells. Although each pathway is governed by its own set of factors, they all contain Rab GTPases that serve as master regulators. In this review, we discuss how Rabs can regulate virtually all steps of membrane traffic from the formation of the transport vesicle at the donor membrane to its fusion at the target membrane. Some of the many regulatory functions performed by Rabs include interacting with diverse effector proteins that select cargo, promoting vesicle movement, and verifying the correct site of fusion. We describe cascade mechanisms that may define directionality in traffic and ensure that different Rabs do not overlap in the pathways that they regulate. Throughout this review we highlight how Rab dysfunction leads to a variety of disease states ranging from infectious diseases to cancer.
PMCID: PMC3710122  PMID: 21248164
7.  GTPase Networks in Membrane Traffic 
Annual review of biochemistry  2012;81:637-659.
Members of the Rab or ARF/Sar branches of the Ras GTPase super-family regulate almost every step of intracellular membrane traffic. A rapidly growing body of evidence indicates that these GTPases do not act as lone agents but are networked to one another through a variety of mechanisms to coordinate the individual events of one stage of transport and to link together the different stages of an entire transport pathway. These mechanisms include guanine nucleotide exchange factor (GEF) cascades, GTPase-activating protein (GAP) cascades, effectors that bind to multiple GTPases, and positive-feedback loops generated by exchange factor-effector interactions. Together these mechanisms can lead to an ordered series of transitions from one GTPase to the next. As each GTPase recruits a unique set of effectors, these transitions help to define changes in the functionality of the membrane compartments with which they are associated.
PMCID: PMC3708692  PMID: 22463690
Rab; ARF; guanine nucleotide exchange factor; GTPase-activating protein; effector; vesicular transport
8.  ER network formation requires a balance of the dynamin-like GTPase Sey1p and Lnp1p, a member of the Lunapark family 
Nature Cell Biology  2012;14(7):707-716.
While studies on endoplasmic reticulum (ER) structure and dynamics have focused on the ER tubule forming proteins (reticulons and DP1/Yop1p) and the tubule fusion protein atlastin, nothing is known about the proteins and processes that act to counter-balance this machinery. Here we show that Lnp1p, a member of the conserved lunapark family, plays a role in ER network formation. Lnp1p binds to the reticulons and Yop1p and resides at ER tubule junctions in both yeast and mammalian cells. In the yeast Saccharomyces cerevisiae, the interaction of Lnp1p with the reticulon protein, Rtn1p, and the localization of Lnp1p to ER junctions are regulated by Sey1p, the yeast ortholog of atlastin. We propose that Lnp1p and Sey1p act antagonistically to balance polygonal network formation. In support of this proposal, we show that the collapsed, densely reticulated ER network in lnp1Δ cells is partially restored when the GTPase activity of Sey1p is abrogated.
PMCID: PMC3389217  PMID: 22729086
9.  The genome of the green anole lizard and a comparative analysis with birds and mammals 
Nature  2011;477(7366):587-591.
The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments1. Among amniotes, genome sequences are available for mammals2 and birds3–5, but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes3. Also, A. carolinensis mobile elements are very young and diverse – more so than in any other sequenced amniote genome. This lizard genome’s GC content is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds6. We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations.
PMCID: PMC3184186  PMID: 21881562
10.  Phosphatidylinositol 4-phosphate controls both membrane recruitment and a regulatory switch of the Rab GEF, Sec2p 
Developmental cell  2010;18(5):828-840.
Sec2p is the guanine nucleotide exchange factor (GEF) that activates the Rab GTPase Sec4p on secretory vesicles. Sec2p also binds a Rab acting earlier in the secretory pathway, Ypt32p-GTP, forming a RabGEF cascade. Ypt32p and the Sec4p effector Sec15p (a component of the exocyst complex) compete for binding to Sec2p. Indeed Ypt32p initially recruits Sec2p, but subsequently allows a handoff of active Sec2p/Sec4p to Sec15p. Intriguingly, Golgi-associated phosphatidylinositol 4-phosphate (PI4P) works together with Ypt32-GTP in this context. PI4P inhibits Sec2p-Sec15p interactions, promoting recruitment of Sec2p by Ypt32p as secretory vesicles form. However, PI4P levels appear to decline as vesicles reach secretory sites, allowing Sec15p to replace Ypt32p as vesicles mature. In this way, the regulation of PI4P levels may switch Sec2p/Sec4p function during vesicle maturation, from a RabGEF recruitment cascade involving Ypt32p, to an effector positive feedback loop involving Sec15p.
PMCID: PMC2877039  PMID: 20493815
11.  Establishing a Role for the GTPase Ypt1p at the Late Golgi 
Traffic (Copenhagen, Denmark)  2010;11(4):520-532.
GTPases of the Rab family cycle between an inactive (GDP-bound) and active (GTP-bound) conformation. The active form of the Rab regulates a variety of cellular functions via multiple effectors. Guanine nucleotide exchange factors (GEFs) activate Rabs by accelerating the exchange of GDP for GTP, while GTPase activating proteins (GAPs) inactivate Rabs by stimulating the hydrolysis of GTP. The GTPase Ypt1p is required for ER-Golgi and intra-Golgi traffic in the yeast Saccharomyces cerevisiae. Recent findings, however, have shown that a Ypt1p GEF, GAP and effector are all required for traffic from the early endosome to the Golgi. Here we describe a screen for ypt1 mutants that block traffic from the early endosome to the late Golgi, but not general secretion. This screen has led to the identification of a collection of recessive and dominant mutants that block traffic from the early endosome. While it has long been known that Ypt1p regulates the flow of biosynthetic traffic into the cis side of the Golgi, these findings have established a role for Ypt1p in the regulation of early endosome-Golgi traffic. We propose that Ypt1p regulates the flow of traffic into the cis and trans side of the Golgi via multiple effectors.
PMCID: PMC2861988  PMID: 20059749
Rab; early endosome; Golgi; membrane traffic
12.  The Evolution and Diversity of DNA Transposons in the Genome of the Lizard Anolis carolinensis 
DNA transposons have considerably affected the size and structure of eukaryotic genomes and have been an important source of evolutionary novelties. In vertebrates, DNA transposons are discontinuously distributed due to the frequent extinction and recolonization of these genomes by active elements. We performed a detailed analysis of the DNA transposons in the genome of the lizard Anolis carolinensis, the first non-avian reptile to have its genome sequenced. Elements belonging to six of the previously recognized superfamilies of elements (hAT, Tc1/Mariner, Helitron, PIF/Harbinger, Polinton/Maverick, and Chapaev) were identified. However, only four (hAT, Tc1/Mariner, Helitron, and Chapaev) of these superfamilies have successfully amplified in the anole genome, producing 67 distinct families. The majority (57/67) are nonautonomous and demonstrate an extraordinary diversity of structure, resulting from frequent interelement recombination and incorporation of extraneous DNA sequences. The age distribution of transposon families differs among superfamilies and reveals different dynamics of amplification. Chapaev is the only superfamily to be extinct and is represented only by old copies. The hAT, Tc1/Mariner, and Helitron superfamilies show different pattern of amplification, yet they are predominantly represented by young families, whereas divergent families are exceedingly rare. Although it is likely that some elements, in particular long ones, are subjected to purifying selection and do not reach fixation, the majority of families are neutral and accumulate in the anole genome in large numbers. We propose that the scarcity of old copies in the anole genome results from the rapid decay of elements, caused by a high rate of DNA loss.
PMCID: PMC3014272  PMID: 21127169
transposon; transposase; recombination; Anolis
13.  Activation of the Mitogen-activated Protein Kinase, Slt2p, at Bud Tips Blocks a Late Stage of Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2010;21(10):1772-1782.
We explore the role of components that act both upstream and downstream of Slt2p in the Ptc1p-dependent regulation of ER inheritance and mitochondrial inheritance. Our findings are that Ptc1p is needed to inactivate the pool of Slt2p associated with the bud tip to promote the cortical distribution of the ER in daughter cells.
Inheritance of the endoplasmic reticulum (ER) requires Ptc1p, a type 2C protein phosphatase of Saccharomyces cerevisiae. Genetic analysis indicates that Ptc1p is needed to inactivate the cell wall integrity (CWI) MAP kinase, Slt2p. Here we show that under normal growth conditions, Ptc1p inactivates Slt2p just as ER tubules begin to spread from the bud tip along the cortex. In ptc1Δ cells, the propagation of cortical ER from the bud tip to the periphery of the bud is delayed by hyperactivation of Slt2p. The pool of Slt2p that controls ER inheritance requires the CWI pathway scaffold, Spa2p, for its retention at the bud tip, and a mutation within Slt2p that prevents its association with the bud tip blocks its role in ER inheritance. These results imply that Slt2p inhibits a late step in ER inheritance by phosphorylating a target at the tip of daughter cells. The PI4P5-kinase, Mss4p, is an upstream activator of this pool of Slt2p. Ptc1p-dependant inactivation of Slt2p is also needed for mitochondrial inheritance; however, in this case, the relevant pool of Slt2p is not at the bud tip.
PMCID: PMC2869382  PMID: 20357006
14.  Analysis of SEC9 Suppression Reveals a Relationship of SNARE Function to Cell Physiology 
PLoS ONE  2009;4(5):e5449.
Growth and division of Saccharomyces cerevisiae is dependent on the action of SNARE proteins that are required for membrane fusion. SNAREs are regulated, through a poorly understood mechanism, to ensure membrane fusion at the correct time and place within a cell. Although fusion of secretory vesicles with the plasma membrane is important for yeast cell growth, the relationship between exocytic SNAREs and cell physiology has not been established.
Methodology/Principal Findings
Using genetic analysis, we identified several influences on the function of exocytic SNAREs. Genetic disruption of the V-ATPase, but not vacuolar proteolysis, can suppress two different temperature-sensitive mutations in SEC9. Suppression is unlikely due to increased SNARE complex formation because increasing SNARE complex formation, through overexpression of SRO7, does not result in suppression. We also observed suppression of sec9 mutations by growth on alkaline media or on a non-fermentable carbon source, conditions associated with a reduced growth rate of wild-type cells and decreased SNARE complex formation.
Three main conclusions arise from our results. First, there is a genetic interaction between SEC9 and the V-ATPase, although it is unlikely that this interaction has functional significance with respect to membrane fusion or SNAREs. Second, Sro7p acts to promote SNARE complex formation. Finally, Sec9p function and SNARE complex formation are tightly coupled to the physiological state of the cell.
PMCID: PMC2674220  PMID: 19421331
15.  An Internal Domain of Exo70p Is Required for Actin-independent Localization and Mediates Assembly of Specific Exocyst Components 
Molecular Biology of the Cell  2009;20(1):153-163.
The exocyst consists of eight rod-shaped subunits that align in a side-by-side manner to tether secretory vesicles to the plasma membrane in preparation for fusion. Two subunits, Sec3p and Exo70p, localize to exocytic sites by an actin-independent pathway, whereas the other six ride on vesicles along actin cables. Here, we demonstrate that three of the four domains of Exo70p are essential for growth. The remaining domain, domain C, is not essential but when deleted, it leads to synthetic lethality with many secretory mutations, defects in exocyst assembly of exocyst components Sec5p and Sec6p, and loss of actin-independent localization. This is analogous to a deletion of the amino-terminal domain of Sec3p, which prevents an interaction with Cdc42p or Rho1p and blocks its actin-independent localization. The two mutations are synthetically lethal, even in the presence of high copy number suppressors that can bypass complete deletions of either single gene. Although domain C binds Rho3p, loss of the Exo70p-Rho3p interaction does not account for the synthetic lethal interactions or the exocyst assembly defects. The results suggest that either Exo70p or Sec3p must associate with the plasma membrane for the exocyst to function as a vesicle tether.
PMCID: PMC2613103  PMID: 18946089
16.  A catalytic coiled-coil: structural insights into the activation of the Rab GTPase Sec4p by Sec2p 
Molecular cell  2007;25(3):455-462.
Rab GTPases, the largest subgroup in the superfamily of Ras-like GTPases, play regulatory roles in multiple steps of intracellular vesicle trafficking. They are activated by guanine nucleotide exchange factors (GEFs), which catalyze the interconversion of the GDP-bound, or inactive, form of Rab to the GTP-bound, or active, form. Relatively little is known of the mechanisms by which GEFs activate Rabs. Here we present the crystal structure of the GEF domain of Sec2p in complex with its Rab partner Sec4p. The Sec2p GEF domain is a 220 Å long coiled-coil, striking in its simplicity and in the use of the coiled-coil motif for catalysis. The structure suggests a mechanism whereby Sec2p induces extensive structural rearrangements in the Sec4p switch regions and phosphate binding loop that are incompatible with nucleotide binding. We show that Sec2p is specific for Sec4p and that specificity determinants reside in the two switch regions of Sec4p.
PMCID: PMC1847580  PMID: 17289591
17.  Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum 
Molecular Biology of the Cell  2006;17(7):3009-3020.
The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.
PMCID: PMC1483037  PMID: 16624861
18.  The rab Exchange Factor Sec2p Reversibly Associates with the Exocyst 
Molecular Biology of the Cell  2006;17(6):2757-2769.
Activation of the rab GTPase, Sec4p, by its exchange factor, Sec2p, is needed for polarized transport of secretory vesicles to exocytic sites and for exocytosis. A small region in the C-terminal half of Sec2p regulates its localization. Loss of this region results in temperature-sensitive growth and the depolarized accumulation of secretory vesicles. Here, we show that Sec2p associates with the exocyst, an octameric effector of Sec4p involved in tethering secretory vesicles to the plasma membrane. Specifically, the exocyst subunit Sec15p directly interacts with Sec2p. This interaction normally occurs on secretory vesicles and serves to couple nucleotide exchange on Sec4p to the recruitment of the Sec4p effector. The mislocalization of Sec2p mutants correlates with dramatically enhanced binding to the exocyst complex. We propose that Sec2p is normally released from the exocyst after vesicle tethering so that it can recycle onto a new round of vesicles. The mislocalization of Sec2p mutants results from a failure to be released from Sec15p, blocking this recycling pathway.
PMCID: PMC1474791  PMID: 16611746
19.  The yeast lgl family member Sro7p is an effector of the secretory Rab GTPase Sec4p 
The Journal of Cell Biology  2006;172(1):55-66.
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.
PMCID: PMC2063532  PMID: 16390997
20.  Functional specialization within a vesicle tethering complex 
The Journal of Cell Biology  2004;167(5):875-887.
The exocyst is an octameric protein complex required to tether secretory vesicles to exocytic sites and to retain ER tubules at the apical tip of budded cells. Unlike the other five exocyst genes, SEC3, SEC5, and EXO70 are not essential for growth or secretion when either the upstream activator rab, Sec4p, or the downstream SNARE-binding component, Sec1p, are overproduced. Analysis of the suppressed sec3Δ, sec5Δ, and exo70Δ strains demonstrates that the corresponding proteins confer differential effects on vesicle targeting and ER inheritance. Sec3p and Sec5p are more critical than Exo70p for ER inheritance. Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled. Sec1p overproduction results in increased SNARE complex levels, indicating a role in assembly or stabilization of SNARE complexes. Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst. Our results suggest that Sec1p couples exocyst-mediated vesicle tethering with SNARE-mediated docking and fusion.
PMCID: PMC2172455  PMID: 15583030
21.  Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p 
The Journal of Cell Biology  2004;167(5):889-901.
Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.
PMCID: PMC2172445  PMID: 15583031
22.  Myo4p and She3p are required for cortical ER inheritance in Saccharomyces cerevisiae 
The Journal of Cell Biology  2003;163(6):1255-1266.
Myo4p is a nonessential type V myosin required for the bud tip localization of ASH1 and IST2 mRNA. These mRNAs associate with Myo4p via the She2p and She3p proteins. She3p is an adaptor protein that links Myo4p to its cargo. She2p binds to ASH1 and IST2 mRNA, while She3p binds to both She2p and Myo4p. Here we show that Myo4p and She3p, but not She2p, are required for the inheritance of cortical ER in the budding yeast Saccharomyces cerevisiae. Consistent with this observation, we find that cortical ER inheritance is independent of mRNA transport. Cortical ER is a dynamic network that forms cytoplasmic tubular connections to the nuclear envelope. ER tubules failed to grow when actin polymerization was blocked with the drug latrunculin A (Lat-A). Additionally, a reduction in the number of cytoplasmic ER tubules was observed in Lat-A–treated and myo4Δ cells. Our results suggest that Myo4p and She3p facilitate the growth and orientation of ER tubules.
PMCID: PMC2173705  PMID: 14691136
cortical ER inheritance; Myo4p; She proteins; myosin; yeast
23.  Sec3p Is Needed for the Spatial Regulation of Secretion and for the Inheritance of the Cortical Endoplasmic ReticulumV⃞ 
Molecular Biology of the Cell  2003;14(12):4770-4782.
Sec3p is a component of the exocyst complex that tethers secretory vesicles to the plasma membrane at exocytic sites in preparation for fusion. Unlike all other exocyst structural genes, SEC3 is not essential for growth. Cells lacking Sec3p grow and secrete surprisingly well at 25°C; however, late markers of secretion, such as the vesicle marker Sec4p and the exocyst subunit Sec8p, localize more diffusely within the bud. Furthermore, sec3Δ cells are strikingly round relative to wild-type cells and are unable to form pointed mating projections in response to α factor. These phenotypes support the proposed role of Sec3p as a spatial landmark for secretion. We also find that cells lacking Sec3p exhibit a dramatic defect in the inheritance of cortical ER into the bud, whereas the inheritance of mitochondria and Golgi is unaffected. Overexpression of Sec3p results in a prominent patch of the endoplasmic reticulum (ER) marker Sec61p-GFP at the bud tip. Cortical ER inheritance in yeast has been suggested to involve the capture of ER tubules at the bud tip. Sec3p may act in this process as a spatial landmark for cortical ER inheritance.
PMCID: PMC284782  PMID: 12960429
24.  Ypt32 recruits the Sec4p guanine nucleotide exchange factor, Sec2p, to secretory vesicles; evidence for a Rab cascade in yeast 
The Journal of Cell Biology  2002;157(6):1005-1016.
SEC2 is an essential gene required for polarized growth of the yeast Saccharomyces cerevisiae. It encodes a protein of 759 amino acids that functions as a guanine nucleotide exchange factor for the small GTPase Sec4p, a regulator of Golgi to plasma membrane transport. Activation of Sec4p by Sec2p is needed for polarized transport of vesicles to exocytic sites. Temperature-sensitive (ts) mutations in sec2 and sec4 result in a tight block in secretion and the accumulation of secretory vesicles randomly distributed in the cell. The proper localization of Sec2p to secretory vesicles is essential for its function and is largely independent of Sec4p. Although the ts mutation sec2-78 does not affect nucleotide exchange activity, the protein is mislocalized. Here we present evidence that Ypt31/32p, members of Rab family of GTPases, regulate Sec2p function. First, YPT31/YPT32 suppress the sec2-78 mutation. Second, overexpression of Ypt31/32p restores localization of Sec2-78p. Third, Ypt32p and Sec2p interact biochemically, but Sec2p has no exchange activity on Ypt32p. We propose that Ypt32p and Sec4p act as part of a signaling cascade in which Ypt32p recruits Sec2p to secretory vesicles; once on the vesicle, Sec2p activates Sec4p, enabling the polarized transport of vesicles to the plasma membrane.
PMCID: PMC2174052  PMID: 12045183
membrane traffic; Ypt31/32; exchange factor; Rab; yeast
25.  Pag1p, a Novel Protein Associated with Protein Kinase Cbk1p, Is Required for Cell Morphogenesis and Proliferation in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2002;13(2):503-514.
Protein kinases in the Cot-1/Orb6/Ndr/Warts family are important regulators of cell morphogenesis and proliferation. Cbk1p, a member of this family in Saccharomyces cerevisiae, has previously been shown to be required for normal morphogenesis in vegetatively growing cells and in haploid cells responding to mating pheromone. A mutant of PAG1, a novel gene in S. cerevisiae, displayed defects similar to those of cbk1 mutants. pag1 and cbk1 mutants share a common set of suppressors, including the disruption of SSD1, a gene encoding an RNA binding protein, and the overexpression of Sim1p, an extracellular protein. These genetic results suggest that PAG1 and CBK1 act in the same pathway. Furthermore, we found that Pag1p and Cbk1p localize to the same polarized peripheral sites and that they coimmunoprecipitate with each other. Pag1p is a conserved protein. The homologs of Pag1p in other organisms are likely to form complexes with the Cbk1p-related kinases and function with those kinases in the same biological processes.
PMCID: PMC65645  PMID: 11854408

Results 1-25 (41)