Interactions between Sla1p, Lsb5p and Arf3p in yeast endocytosis

2005 ◽  
Vol 33 (6) ◽  
pp. 1273-1275 ◽  
Author(s):  
R. Costa ◽  
K.R. Ayscough
Keyword(s):  
Actin Cytoskeleton ◽  
Mammalian Cells ◽  
Cell Signalling ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Pheromone Receptor ◽  
Cargo Proteins ◽  
Endocytic Machinery ◽  
Adp Ribosylation Factor ◽  
Uptake Of Nutrients

Endocytosis is critical for controlling the protein–lipid composition of the plasma membrane, uptake of nutrients as well as pathogens, and also plays an important role in regulation of cell signalling. While a number of pathways for endocytosis have been characterized in different organisms, all of these require remodelling of the cell cortex. The importance of a dynamic actin cytoskeleton for facilitating endocytosis has been recognized for many years in budding yeast, and is increasingly supported by studies in mammalian cells. Our studies have focused on proteins that we have shown to act at the interface between the actin cytoskeleton and the endocytic machinery. In particular, we have studied interactions of Sla1p, which binds to both activators of actin dynamics, i.e. Abp1p, Las17p and Pan1p, and to cargo proteins such as the pheromone receptor Ste2p. More recently we have mapped the interaction of Sla1p with Lsb5p, a protein that has a similar structure to the GGA [Golgi-localizing, γ-adaptin ear homology domain, Arf (ADP-ribosylation factor)-binding] family of proteins with an N-terminal VHS (Vps27p/Hrs/STAM)-domain and a GAT (GGAs and TOM1) domain. We show that Lsb5p can interact with yeast Arf3p (orthologous with mammalian Arf6) and we demonstrate a requirement for Arf3p expression in order to localize Lsb5p to the cell cortex.

scholarly journals Lsb5p interacts with actin regulators Sla1p and Las17p, ubiquitin and Arf3p to couple actin dynamics to membrane trafficking processes

2005 ◽  
Vol 387 (3) ◽  
pp. 649-658 ◽  
Author(s):  
Rosaria COSTA ◽  
Derek T. WARREN ◽  
Kathryn R. AYSCOUGH
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Active Form ◽  
Actin Dynamics ◽  
Binding Motif ◽  
Pheromone Receptor ◽  
Cargo Proteins ◽  
Endocytic Machinery ◽  
Vacuolar Protein ◽  
First Time

The importance of coupling the process of endocytosis to factors that regulate actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified. Sla1p is a well-characterized yeast protein that binds both to activators of actin dynamics, Las17p and Pan1p, and to cargo proteins, such as the pheromone receptor Ste2p. Previously, we reported that the Lsb5 protein plays a role in endocytosis in yeast and that it localizes to the plasma membrane. Lsb5p has a similar structure to the GGA [Golgi-localized, γ-ear-containing, Arf (ADP-ribosylation factor)-binding] family of proteins with an N-terminal VHS [Vps27p (vacuolar protein sorting protein 27), Hrs, Stam] domain and a GAT (GGA and Tom1) domain. It does not, however, contain either a γ-adaptin ear or a clathrin-binding motif. In the present study, we have further defined its interaction site with both Sla1p and with Las17p, two regulators of actin dynamics. The site of interaction with Sla1p involves the Sla1 HD1 (homology domain 1), which also was shown previously to interact with the pheromone receptor Ste2p. We also demonstrate hitherto unknown interactions between Lsb5p and the active form of the yeast Arf3 protein, and with ubiquitin. Finally, we demonstrate a requirement for Arf3p expression in order to localize Lsb5p to the correct cortical site in cells. Taken together, our data provide further evidence for the role of Lsb5p in membrane-trafficking events at the plasma membrane and also demonstrate for the first time an interaction of Arf3 with the endocytic machinery in yeast.

scholarly journals Novel Proteins Linking the Actin Cytoskeleton to the Endocytic Machinery in Saccharomyces cerevisiae

2002 ◽  
Vol 13 (10) ◽  
pp. 3646-3661 ◽  
Author(s):  
H. Dewar ◽  
D. T. Warren ◽  
F. C. Gardiner ◽  
C. G. Gourlay ◽  
N. Satish ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Elevated Temperatures ◽  
Adaptor Protein ◽  
Fluid Phase ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Fluid Phase Endocytosis ◽  
Endocytic Machinery

The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.

Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics

2002 ◽  
Vol 115 (8) ◽  
pp. 1703-1715 ◽  
Author(s):  
Derek T. Warren ◽  
Paul D. Andrews ◽  
Campbell W. Gourlay ◽  
Kathryn R. Ayscough
Keyword(s):  
Immunofluorescence Microscopy ◽  
Fluid Phase ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Binding Studies ◽  
Endocytic Machinery ◽  
Single Complex ◽  
Actin Patch ◽  
First Time

Sla1p is a protein required for cortical actin patch structure and organisation in budding yeast. Here we use a combination of immunofluorescence microscopy and biochemical approaches to demonstrate interactions of Sla1p both with proteins regulating actin dynamics and with proteins required for endocytosis. Using Sla1p-binding studies we reveal association of Sla1p with two proteins known to be important for activation of the Arp2/3 complex in yeast, Abp1p and the yeast WASP homologue Las17p/Bee1p. A recent report of Sla1p association with Pan1p puts Sla1p in the currently unique position of being the only yeast protein known to interact with all three known Arp2/3-activating proteins in yeast. Localisation of Sla1p at the cell cortex is, however, dependent on the EH-domain-containing protein End3p, which is part of the yeast endocytic machinery. Using spectral variants of GFP on Sla1p(YFP) and on Abp1p (CFP) we show for the first time that these proteins can exist in discrete complexes at the cell cortex. However, the detection of a significant FRET signal means that these proteins also come close together in a single complex, and it is in this larger complex that we propose that Sla1p binding to Abp1p and Las17p/Bee1p is able to link actin dynamics to the endocytic machinery. Finally, we demonstrate marked defects in both fluid-phase and receptor-mediated endocytosis in cells that do not express SLA1, indicating that Sla1p is central to the requirement in yeast to couple endocytosis with the actin cytoskeleton.

scholarly journals Insights into the evolution of regulated actin dynamics via characterization of primitive gelsolin/cofilin proteins from Asgard archaea

2020 ◽  
Vol 117 (33) ◽  
pp. 19904-19913 ◽  
Author(s):  
Caner Akıl ◽  
Linh T. Tran ◽  
Magali Orhant-Prioux ◽  
Yohendran Baskaran ◽  
Edward Manser ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Calcium Binding ◽  
Mammalian Cells ◽  
Actin Polymerization ◽  
Calcium Release ◽  
Duplication Event ◽  
Cellular Level ◽  
Actin Dynamics

Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures.

scholarly journals Gap43, Marcks, and Cap23 Modulate Pi(4,5)p2 at Plasmalemmal Rafts, and Regulate Cell Cortex Actin Dynamics through a Common Mechanism

2000 ◽  
Vol 149 (7) ◽  
pp. 1455-1472 ◽  
Author(s):  
Thorsten Laux ◽  
Kiyoko Fukami ◽  
Marcus Thelen ◽  
Tamara Golub ◽  
Dunja Frey ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Dynamic Properties ◽  
Peripheral Nerve Regeneration ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Common Mechanism ◽  
Cell Cortex ◽  
Kinase Substrate ◽  
Nerve Sprouting ◽  
Process Formation

The dynamic properties of the cell cortex and its actin cytoskeleton determine important aspects of cell behavior and are a major target of cell regulation. GAP43, myristoylated alanine-rich C kinase substrate (MARCKS), and CAP23 (GMC) are locally abundant, plasmalemma-associated PKC substrates that affect actin cytoskeleton. Their expression correlates with morphogenic processes and cell motility, but their role in cortex regulation has been difficult to define mechanistically. We now show that the three proteins accumulate at rafts, where they codistribute with PI(4,5)P2, and promote its retention and clustering. Binding and modulation of PI(4,5)P2 depended on the basic effector domain (ED) of these proteins, and constructs lacking the ED functioned as dominant inhibitors of plasmalemmal PI(4,5)P2 modulation. In the neuronlike cell line, PC12, NGF- and substrate-induced peripheral actin structures, and neurite outgrowth were greatly augmented by any of the three proteins, and suppressed by ΔED mutants. Agents that globally mask PI(4,5)P2 mimicked the effects of GMC on peripheral actin recruitment and cell spreading, but interfered with polarization and process formation. Dominant negative GAP43(ΔED) also interfered with peripheral nerve regeneration, stimulus-induced nerve sprouting and control of anatomical plasticity at the neuromuscular junction of transgenic mice. These results suggest that GMC are functionally and mechanistically related PI(4,5)P2 modulating proteins, upstream of actin and cell cortex dynamics regulation.

scholarly journals Actin dynamics in living mammalian cells

1998 ◽  
Vol 111 (12) ◽  
pp. 1649-1658 ◽  
Author(s):  
C. Ballestrem ◽  
B. Wehrle-Haller ◽  
B.A. Imhof
Keyword(s):  
Actin Cytoskeleton ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Time Lapse ◽  
Living Cells ◽  
Actin Dynamics ◽  
Mammalian Cell Lines ◽  
Cytoskeletal Dynamics ◽  
Green Fluorescent

The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human β-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured ‘actin clouds’ were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.

scholarly journals The IQGAP-related protein DGAP1 interacts with Rac and is involved in the modulation of the F-actin cytoskeleton and control of cell motility

1998 ◽  
Vol 111 (20) ◽  
pp. 3059-3071 ◽  
Author(s):  
J. Faix ◽  
C. Clougherty ◽  
A. Konzok ◽  
U. Mintert ◽  
J. Murphy ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Motility ◽  
Fluorescent Protein ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
A Cell ◽  
Microfilament System ◽  
Green Fluorescent ◽  
And Control

DGAP1 of Dictyostelium discoideum is a cell cortex associated 95 kDa protein that shows homology to both RasGTPase-activating proteins (RasGAPs) and RasGAP-related proteins. When tested for RasGAP activity, recombinant DGAP1 protein did not promote the GTPase activity of human H-Ras or of Dictyostelium RasG in vitro. Instead, DGAP1 bound to Dictyostelium Rac1A and human Rac1, but not to human Cdc42. DGAP1 preferentially interacted with the activated GTP-bound forms of Rac1 and Rac1A, but did not affect the GTPase activities. Since Rho-type GTPases are implicated in the formation of specific F-actin structures and in the control of cell morphology, the microfilament system of mutants that either lack or overexpress DGAP1 has been analysed. DGAP1-null mutants showed elevated levels of F-actin that was organised in large leading edges, membrane ruffles or numerous large filopods. Expression of actin fused to green fluorescent protein (GFP) was used to monitor the actin dynamics in these cells, and revealed that the F-actin cytoskeleton of DGAP1-null cells was rapidly re-arranged to form ruffles and filopods. Conversely, in DGAP1-overexpressing cells, the formation of cellular projections containing F-actin was largely suppressed. Measurement of cell migration demonstrated that DGAP1 expression is inversely correlated with the speed of cell motility.

scholarly journals Regulation of the Actin Cytoskeleton via Rho GTPase Signalling in Dictyostelium and Mammalian Cells: A Parallel Slalom

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1592
Author(s):  
Vedrana Filić ◽  
Lucija Mijanović ◽  
Darija Putar ◽  
Antea Talajić ◽  
Helena Ćetković ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Mammalian Cells ◽  
Large Scale ◽  
Rho Gtpase ◽  
Small Gtpases ◽  
Actin Dynamics ◽  
Cellular Motility ◽  
Binary Division ◽  
Cytoskeleton Dynamics

Both Dictyostelium amoebae and mammalian cells are endowed with an elaborate actin cytoskeleton that enables them to perform a multitude of tasks essential for survival. Although these organisms diverged more than a billion years ago, their cells share the capability of chemotactic migration, large-scale endocytosis, binary division effected by actomyosin contraction, and various types of adhesions to other cells and to the extracellular environment. The composition and dynamics of the transient actin-based structures that are engaged in these processes are also astonishingly similar in these evolutionary distant organisms. The question arises whether this remarkable resemblance in the cellular motility hardware is accompanied by a similar correspondence in matching software, the signalling networks that govern the assembly of the actin cytoskeleton. Small GTPases from the Rho family play pivotal roles in the control of the actin cytoskeleton dynamics. Indicatively, Dictyostelium matches mammals in the number of these proteins. We give an overview of the Rho signalling pathways that regulate the actin dynamics in Dictyostelium and compare them with similar signalling networks in mammals. We also provide a phylogeny of Rho GTPases in Amoebozoa, which shows a variability of the Rho inventories across different clades found also in Metazoa.

scholarly journals A Role for the Actin Cytoskeleton of Saccharomyces cerevisiae in Bipolar Bud-Site Selection

1997 ◽  
Vol 136 (1) ◽  
pp. 111-123 ◽  
Author(s):  
Shirley Yang ◽  
Kathryn R. Ayscough ◽  
David G. Drubin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Site Selection ◽  
Mitotic Checkpoint ◽  
Cell Cortex ◽  
Spatial Cues ◽  
Genes Encoding ◽  
Mother And Daughter ◽  
Mother Cells ◽  
Bud Site

Saccharomyces cerevisiae cells select bud sites according to one of two predetermined patterns. MATa and MATα cells bud in an axial pattern, and MATa/α cells bud in a bipolar pattern. These budding patterns are thought to depend on the placement of spatial cues at specific sites in the cell cortex. Because cytoskeletal elements play a role in organizing the cytoplasm and establishing distinct plasma membrane domains, they are well suited for positioning bud-site selection cues. Indeed, the septin-containing neck filaments are crucial for establishing the axial budding pattern characteristic of MATa and MATα cells. In this study, we determined the budding patterns of cells carrying mutations in the actin gene or in genes encoding actin-associated proteins: MATa/α cells were defective in the bipolar budding pattern, but MATa and MATα cells still exhibit a normal axial budding pattern. We also observed that MATa/α actin cytoskeleton mutant daughter cells correctly position their first bud at the distal pole of the cell, but mother cells position their buds randomly. The actin cytoskeleton therefore functions in generation of the bipolar budding pattern and is required specifically for proper selection of bud sites in mother MATa/α cells. These observations and the results of double mutant studies support the conclusion that different rules govern bud-site selection in mother and daughter MATa/α cells. A defective bipolar budding pattern did not preclude an sla2-6 mutant from undergoing pseudohyphal growth, highlighting the central role of daughter cell bud-site selection cues in the formation of pseudohyphae. Finally, by examining the budding patterns of mad2-1 mitotic checkpoint mutants treated with benomyl to depolymerize their microtubules, we confirmed and extended previous evidence indicating that microtubules do not function in axial or bipolar bud-site selection.

scholarly journals ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization

2002 ◽  
Vol 13 (9) ◽  
pp. 3078-3095 ◽  
Author(s):  
Annette L. Boman ◽  
Paul D. Salo ◽  
Melissa J. Hauglund ◽  
Nicole L. Strand ◽  
Shelly J. Rensink ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Carboxypeptidase Y ◽  
Minor Role ◽  
Temperature Sensitive ◽  
Adp Ribosylation ◽  
And Function ◽  
Adp Ribosylation Factor

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

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