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First published online 6 May 2003
doi: 10.1242/jcs.00454

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An interaction between Sla1p and Sla2p plays a role in regulating actin dynamics and endocytosis in budding yeast

Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK

View larger version (28K): [in a new window]   Fig. 1. Sla1118-511p is expressed in cells and localises to the cell cortex. (A) Schematic diagram of Sla1p and Sla2p showing domain structure and regions of Sla1p thought to be important for its role in regulating the actin cytoskeleton. (B) Cells expressing full-length Sla1p, Sla1(118-511)p or cells in which sla1 was deleted (KAY302, 351 or 97 respectively) were grown overnight and cell extracts made. Proteins were separated by SDS-PAGE and western blotted using antibodies against Sla1p. As shown, proteins of the appropriate size were found in cells expressing Sla1p. No band was observed in the extract from sla1 cells. As a loading control the same blots were probed with antibodies to a nuclear protein Anc1p. This showed equivalent levels in all lanes. (C) Log phase KAY303 and KAY367 cells expressing Sla1-myc and Sla1(118-511)myc were grown to log phase and processed for immunofluorescence microscopy. Both Sla1p-myc and Sla1(118-511)-myc localise to punctate patches at the cell cortex demonstrating that the parts of Sla1p required for cortical localisation are located elsewhere in the protein. Bar, 5 µM.

View larger version (64K): [in a new window]   Fig. 5. Localisation of Sla1p and Sla2p in wild-type and mutant cells. (A) KAY303 cells expressing Sla2p and Sla1-myc were grown to log phase and processed for immunofluorescence microscopy as described in Materials and Methods. Both Sla1p-myc-containing and Sla2p-containing patches were polarised to the site of bud emergence, to the enlarging bud and to the bud neck. Furthermore, Sla1p and Sla2p showed substantial co-localisation throughout the cell cycle. However, as indicated by arrows, it was also apparent that a number of cortical patches contained only Sla1p-myc, which suggests that the colocalisation was not always complete. Bar, 10 µM. (B) KAY302 (wildtype) and KAY351 (sla1118-511) cells were grown to log phase and then fixed and processed for immunofluorescence microscopy as described in Materials and Methods. Actin was stained using rhodamine-phalloidin, and Sla2p was localised using antibodies raised to the protein. Bar, 10 µM. (C) The distribution of Sla1-myc in an sla2-null background was examined using immunofluorescence microscopy. In wild-type cells Sla1p was localised to areas of active cell growth, whereas in the absence of sla2 expression Sla1p distribution was depolarised and Sla1p-containing patches appeared evenly distributed around the mother cell and bud. Co-staining with rhodamine-phalloidin revealed that Sla1p-myc was still localised to a subset of cortical patches that contained actin. These images also suggested that in cells lacking Sla2p there are novel structures containing distinct regions of Sla1p and actin organisation (arrows). Bars, 10 µM. The inset shows part of the same image magnified two-fold, and the arrow points toward the same structure in both figures.

View larger version (61K): [in a new window]   Fig. 2. Sla1p interacts with Sla2p in a two-hybrid screen. A two-hybrid screen was performed as described in Materials and Methods with a bait plasmid carrying the amino acid 118-511 region of Sla1p. The minimum interacting sequence was between residues 310 and 768 of Sla2p. Two-hybrid interactions between a number of different Sla1 and Sla2 constructs were tested, as shown. (A) Summary of domain interactions. The strength of interaction was determined by the extent of growth after 5 days with +++ denoting the greatest growth. Corresponding to positions on this table are strains which in B are growing on synthetic medium lacking uracil and leucine, indicating that both plasmids are present, and in C are growing on synthetic medium lacking histidine, adenine, uracil and leucine, indicating that a positive interaction between Sla1p and Sla2p has taken place.

View larger version (19K): [in a new window]   Fig. 3. Sla1p and Sla2p interact in vitro. (A) GST-Sla1p was purified as described in Materials and Methods. Cell extracts were passed over the protein on a column and proteins in bound and unbound fractions were separated by SDS-PAGE and transferred to PVDF for analysis. Western blots were probed with antibodies raised against Sla2p. Sla2p bound only to Sla1p carrying beads and not to control beads alone. FT, flow through; W1,W3, washes 1 and 3; B, bound. (B) To investigate whether the interaction between Sla1p and Sla2p was direct, His-tagged Sla2p was purified from cells as described in Materials and Methods, and its binding to GST control beads or to GST-Sla1p beads was assessed. As shown in the upper panel Sla2p only binds to Sla1p-containing beads. As a control, the extracts from KAY419 cells from which GST-Sla1p was purified were probed to demonstrate the presence of Sla2p in the extract. Then, following GST-Sla1p purification the beads were probed to demonstrate that no Sla2p had co-purified with the Sla1p. Therefore, the Sla1p-Sla2p binding observed was only caused by the presence of the added Sla2p to the GST-Sla1p beads. FT, flow through; W1,W3, washes 1 and 3; B, bound. (C) Bacterially expressed GST or GST-Sla1(118-511SH3#3) was incubated with His-tagged Sla2p as described in Materials and Methods. The beads were washed and the input material (I), wash (W) and bound (B) fractions were run on a gel that was then stained using Coomassie dye. The three bands marked x are all detected by western blotting with GST antibodies indicating that they are all Sla1 degradation products; bands marked were detectable using Sla2 antibodies.

View larger version (36K): [in a new window]   Fig. 4. Sucrose density gradient fractionation of wild-type and sla1 cells. Extracts from KAY302 (wildtype) and KAY300 (sla1) cells were prepared and separated on 3-30% sucrose gradients as described in Materials and Methods. Fractions were then run on SDS-PAGE and blotted onto PVDF membranes. The membrane was cut and probed with individual antibodies. Antibodies were used as described in Materials and Methods. Quantitation of band intensity was performed using NIH Image 1.6.1 Software. The y-axis of the graphs is the band intensity in arbitrary units normalised in each case for background. The gradients were repeated three times. Shown here is a representative example.

View larger version (79K): [in a new window]   Fig. 6. Overexpression of Sla1(118-511) region disrupts fluid phase uptake of lucifer yellow and subsequent trafficking to the vacuole. Cells expressing GST alone (KAY439), GST-Sla1(118-511SH3#3) (KAY508), GST-Sla1(118-511SH3#3)* (KAY657) or GST-Sla1 (KAY439) were grown overnight in synthetic media containing 2% glucose, then inoculated into media with 2% galactose to induce expression of the various fusion proteins respectively. (A) Cells expressing GST alone displayed normal lucifer yellow uptake before and after induction and exhibited a strong fluorescence within the vacuole. (B) In cells induced to overexpress GST-Sla1p (118-511SH3#3), lucifer yellow uptake was disrupted. These cells showed little vacuolar staining compared to those expressing GST alone and possessed more abundant brightly stained dots outside the vacuole. Lucifer yellow staining at the plasma membrane of these cells also appeared brighter than in the control cells. (C) A mutant form of Sla1 (118-511SH3#3)* was expressed that cannot bind to another strong interactor of the 118-511 domain (Ysc84p). The same defect in uptake and subsequent trafficking was seen with this mutants with the Sla1(118-511SH3#3) mutant, indicating that the defect is not because of the domain blocking normal Ysc84 function. (D) Overexpression of full-length Sla1p abrogated lucifer yellow uptake but did not result in accumulation of stained endosomes. Bar, 10 µM.

View larger version (62K): [in a new window]   Fig. 7. Rvs167p localises with actin and not Sla2p in sla1 cells. KAY302 (wild-type) and KAY 97 (sla1) cells were processed for immunofluorescence microscopy as described in Materials and Methods. Actin was stained using rhodamine-phalloidin, and Rvs167p was localised using antibodies raised against the protein. Although actin organisation is altered in the sla1 strain, Rvs167p still localises to these aberrant structures, which suggests that its association with actin is not mediated by Sla1p. Bar, 10 µM.

View larger version (58K): [in a new window]   Fig. 8. The actin phenotypes of sla1sla2 cells. (A) Sensitivity to the actin monomer binding drug LAT-A is proposed to reflect the level of actin monomer in cells and therefore can be used to assess to whether a protein increases or decreases the stability of F-actin. Sensitivity to LAT-A was assessed by halo assay as described in Materials and Methods. These results are summarised graphically. Deletion of sla1 reduces the sensitivity to LAT-A whereas sla2 cells are more sensitive to the effects of the drug. The double mutant strains displayed an intermediate phenotype. The data shown are the average of three halo assays. Strains tested were KAY302, KAY97, KAY136 and KAY128. (B) Rhodamine-phalloidin was used to stain the F-actin of wild-type (KAY302), sla1 (KAY97), sla2 (KAY136) and sla1sla2 (KAY128) cells. In the absence of both sla1p and Sla2p, actin is largely localised to the distal pole of the cell. However, in these cells small, less bright patches could often be seen at polarised regions of the cell (arrows), and actin was also seen at the cytokinetic ring (arrowheads) but this represented a small fraction of the actin that was visualised. Bar, 10 µm. To ascertain whether this larger aggregation of actin patches was likely to be functional, the localisation of two F-actin-binding proteins was determined. Both Sac6p (C) and Abp1p (D) were also observed to localise to the distal regions of the cell, indicating that the actin in these structures is associated with a normal complement of actin-binding proteins. Bar, 10 µm.

View larger version (80K): [in a new window]   Fig. 9. Endocytosis and membrane trafficking in sla1sla2 cells. (A) Fluid phase endocytosis was monitored by following uptake of the fluorescent dye lucifer yellow (see Materials and Methods). Wild-type cells show uptake of the dye into vacuoles. Over the same time period sla1 cells show a reduction in uptake whereas sla2 and the sla1sla2 cells show little or no detectable internalisation of the dye. Bar, 10 µm. The percentage of cells showing detectable uptake of the dye into vacuoles is summarised graphically in B. 200 cells were counted in each of three experiments. Error bars represent standard error. (C) Wild-type (D) sla1, (E) sla2 and (F) sla1sla2 cells were processed for electron microscopy using potassium permanganate as described in Materials and Methods. Bar, 5 µm.

View larger version (18K): [in a new window]   Fig. 10. Model depicting interactions of Sla1p and Sla2p at the cell cortex. (1) Sla2p associates at the cell cortex with clathrin light chain and also potentially directly with the plasma membrane through its ENTH domain. (2) The central region of Sla2p is able to recruit Sla1p (through an interaction with a region encompassed by residues 118-361 of Sla1p) and the associated endocytic machinery Pan1p/End3p. (3) Sla1p also binds to the actin-associated proteins Abp1p and Las17p/Bee1p, allowing recruitment of actin patches to these endocytic sites These interactions then lead to the reorganisation of the actin cytoskeleton, which is likely to promote endocytosis. Evidence for these is from these studies: 1, Henry et al. (Henry et al., 2002); 2, this study; 3, Duncan et al., Li and Warren et al. (Duncan et al., 2001; Li, 1997; Warren et al., 2002).

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