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An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited.

Aghamohammadzadeh S, Smaczynska-de Rooij II, Ayscough KR - PLoS ONE (2014)

Bottom Line: This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function.This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites.These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.

ABSTRACT
Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the 'classic' pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the 'classic' pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.

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The Effect of Abp1 mutations on Bulk Endocytosis.(A) Schematic of full length Abp1 and the different mutants analysed in this study. (B) Wild type, abp1Δ and abp1Δ cells expressing wild type ABP1 or mutant versions were grown to mid log phase before treating with Lat-A and following uptake of FM4-64. Cells were classified according to the most prominent staining after 20 minutes FM4-64 incubation. Error bars are standard deviation. (C) The effect of Abp1 mutations on actin organization as assessed by rhodamine phalloidin staining. (D) A number of proteins have been shown to bind to the SH3 domain of Abp1. Deletions in genes encoding 3 of these proteins, Ark1, Prk1 and Srv2/CAP were analysed to determine whether any of these interactions is potentially required for the Abp1 dependent endocytic pathway in the presence of 25 µM Lat-A. Cells were classified according to the most prominent staining after 20 minutes FM4-64 incubation. Error is Std Deviation.
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pone-0103311-g005: The Effect of Abp1 mutations on Bulk Endocytosis.(A) Schematic of full length Abp1 and the different mutants analysed in this study. (B) Wild type, abp1Δ and abp1Δ cells expressing wild type ABP1 or mutant versions were grown to mid log phase before treating with Lat-A and following uptake of FM4-64. Cells were classified according to the most prominent staining after 20 minutes FM4-64 incubation. Error bars are standard deviation. (C) The effect of Abp1 mutations on actin organization as assessed by rhodamine phalloidin staining. (D) A number of proteins have been shown to bind to the SH3 domain of Abp1. Deletions in genes encoding 3 of these proteins, Ark1, Prk1 and Srv2/CAP were analysed to determine whether any of these interactions is potentially required for the Abp1 dependent endocytic pathway in the presence of 25 µM Lat-A. Cells were classified according to the most prominent staining after 20 minutes FM4-64 incubation. Error is Std Deviation.

Mentions: To determine whether specific regions of Abp1 are required for its function in bulk endocytosis mutations were obtained or generated (Figure 5A). Abp1 has 2 acidic domains (N* and C*) previously characterized by Goode and colleagues as defective in Arp2/3 interaction but functional in actin binding. Three mutant forms of Abp1 (N*, C* and both N*C*) were kindly shared by D. Drubin (U.C. Berkeley) for this analysis [31]. In addition Abp1 has a C-terminal SH3 domain demonstrated to bind Ark1, Prk1, Scp1 and Srv2/CAP at endocytic sites [32], [33]. An Abp1 truncation lacking the SH3 domain was also generated. Cells lacking abp1 were transformed with a plasmid carrying wild type or mutant abp1. ABP1 was expressed in these cells under its own promoter on a centromere bearing plasmid. The effect of Abp1 mutants on actin organization was first analysed by rhodamine phalloidin staining to determine whether any defects could be visualized as a result of these mutations (Figure 5B). In all cases cells appeared to have relatively wild type characteristics and organization of both actin patches and cables. The effect of the Abp1 mutants on FM4-64 uptake in the presence or absence of 25 µM Lat-A was then analysed (Figure 5C). As expected in the absence of Lat-A most cells are able to endocytose FM4-64 at a similar level to wild type cells. The slight reduction in endosome to vacuole trafficking in the presence of the abpl1ΔSH3 mutant suggests that this mutation is causing a dominant effect at a stage beyond initial endocytosis. In the presence of Lat-A, only wild type and abp1 cells transformed with ABP1 showed clear FM4-64 uptake. Two mutants caused a very severe inhibition of uptake, these were Abp1 N* and Abp1 N*C* indicating the importance of the N terminal acidic site and the function of Arp2/3 for this endocytic route. The SH3 domain truncation also showed a severe defect, similar to the complete deletion supporting a role for SH3 binding interactions for Abp1 function in this endocytic pathway.


An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited.

Aghamohammadzadeh S, Smaczynska-de Rooij II, Ayscough KR - PLoS ONE (2014)

The Effect of Abp1 mutations on Bulk Endocytosis.(A) Schematic of full length Abp1 and the different mutants analysed in this study. (B) Wild type, abp1Δ and abp1Δ cells expressing wild type ABP1 or mutant versions were grown to mid log phase before treating with Lat-A and following uptake of FM4-64. Cells were classified according to the most prominent staining after 20 minutes FM4-64 incubation. Error bars are standard deviation. (C) The effect of Abp1 mutations on actin organization as assessed by rhodamine phalloidin staining. (D) A number of proteins have been shown to bind to the SH3 domain of Abp1. Deletions in genes encoding 3 of these proteins, Ark1, Prk1 and Srv2/CAP were analysed to determine whether any of these interactions is potentially required for the Abp1 dependent endocytic pathway in the presence of 25 µM Lat-A. Cells were classified according to the most prominent staining after 20 minutes FM4-64 incubation. Error is Std Deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4114835&req=5

pone-0103311-g005: The Effect of Abp1 mutations on Bulk Endocytosis.(A) Schematic of full length Abp1 and the different mutants analysed in this study. (B) Wild type, abp1Δ and abp1Δ cells expressing wild type ABP1 or mutant versions were grown to mid log phase before treating with Lat-A and following uptake of FM4-64. Cells were classified according to the most prominent staining after 20 minutes FM4-64 incubation. Error bars are standard deviation. (C) The effect of Abp1 mutations on actin organization as assessed by rhodamine phalloidin staining. (D) A number of proteins have been shown to bind to the SH3 domain of Abp1. Deletions in genes encoding 3 of these proteins, Ark1, Prk1 and Srv2/CAP were analysed to determine whether any of these interactions is potentially required for the Abp1 dependent endocytic pathway in the presence of 25 µM Lat-A. Cells were classified according to the most prominent staining after 20 minutes FM4-64 incubation. Error is Std Deviation.
Mentions: To determine whether specific regions of Abp1 are required for its function in bulk endocytosis mutations were obtained or generated (Figure 5A). Abp1 has 2 acidic domains (N* and C*) previously characterized by Goode and colleagues as defective in Arp2/3 interaction but functional in actin binding. Three mutant forms of Abp1 (N*, C* and both N*C*) were kindly shared by D. Drubin (U.C. Berkeley) for this analysis [31]. In addition Abp1 has a C-terminal SH3 domain demonstrated to bind Ark1, Prk1, Scp1 and Srv2/CAP at endocytic sites [32], [33]. An Abp1 truncation lacking the SH3 domain was also generated. Cells lacking abp1 were transformed with a plasmid carrying wild type or mutant abp1. ABP1 was expressed in these cells under its own promoter on a centromere bearing plasmid. The effect of Abp1 mutants on actin organization was first analysed by rhodamine phalloidin staining to determine whether any defects could be visualized as a result of these mutations (Figure 5B). In all cases cells appeared to have relatively wild type characteristics and organization of both actin patches and cables. The effect of the Abp1 mutants on FM4-64 uptake in the presence or absence of 25 µM Lat-A was then analysed (Figure 5C). As expected in the absence of Lat-A most cells are able to endocytose FM4-64 at a similar level to wild type cells. The slight reduction in endosome to vacuole trafficking in the presence of the abpl1ΔSH3 mutant suggests that this mutation is causing a dominant effect at a stage beyond initial endocytosis. In the presence of Lat-A, only wild type and abp1 cells transformed with ABP1 showed clear FM4-64 uptake. Two mutants caused a very severe inhibition of uptake, these were Abp1 N* and Abp1 N*C* indicating the importance of the N terminal acidic site and the function of Arp2/3 for this endocytic route. The SH3 domain truncation also showed a severe defect, similar to the complete deletion supporting a role for SH3 binding interactions for Abp1 function in this endocytic pathway.

Bottom Line: This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function.This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites.These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.

ABSTRACT
Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the 'classic' pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the 'classic' pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.

Show MeSH
Related in: MedlinePlus