The dynamin Vps1 mediates Atg9 transport to the sites of autophagosome formation

Autophagy is a key process in eukaryotes to maintain cellular homeostasis by delivering cellular components to lysosomes/vacuoles for degradation and reuse of the resulting metabolites. Membrane rearrangements and trafficking events are mediated by the core machinery of autophagy-related (Atg) proteins, which carry out a variety of functions. How Atg9, a lipid scramblase and the only conserved transmembrane protein within this core Atg machinery, is trafficked during autophagy remained largely unclear. Here, we addressed this question in yeast Saccharomyces cerevisiae and found that retromer complex and dynamin Vps1 mutants alter Atg9 subcellular distribution and severely impair the autophagic flux by affecting two separate autophagy steps. We provide evidence that Vps1 interacts with Atg9 at Atg9 reservoirs. In the absence of Vps1, Atg9 fails to reach the sites of autophagosome formation, and this results in an autophagy defect. The function of Vps1 in autophagy requires its GTPase activity. Moreover, Vps1 point mutants associated with human diseases such as microcytic anemia and Charcot-Marie-Tooth are unable to sustain autophagy and affect Atg9 trafficking. Together, our data provide novel insights on the role of dynamins in Atg9 trafficking and suggest that a defect in this autophagy step could contribute to severe human pathologies.</p

An Abp1-Dependent Route of Endocytosis Functions when the Classical Endocytic Pathway in Yeast Is Inhibited

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

A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic

BACKGROUND: RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation. METHODOLOGY/PRINCIPAL FINDINGS: Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149) that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment. CONCLUSIONS/SIGNIFICANCE: A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Alternative endocytic components and routes in wild type cells.

<p>(A) Cells co-expressing Sla1-mRFP and Sla2-GFP (KAY1734) were imaged and composition of spots was analysed. Kymographs show examples of puncta that show the two proteins together and with Sla2-GFP alone. Kymographs are from full movie (120 seconds) (B) Cells expressing Sla1-GFP (KAY733) were incubated with FM4-64FX for 5 minutes and imaged. Examples of both GFP only and FM4-64 only puncta invaginating are shown in kymographs. Kymographs are from full movie (120 seconds). The images are stills taken from supplemental movie at the times indicated. Scale bar = 2 µm.</p

The effect of abp1 deletion on the uptake and trafficking of Lucifer yellow.

<p>(A) Wild type and <i>abp1</i>Δ cells were grown to mid log phase. Half of the cells were incubated with 25 µM Lat-A. LY was added for 90 minutes as described. Localization of stain was categorized as being at the plasma membrane, endosomes or vacuoles. Error bars are std deviation. (B) Representative images from the cells visualized. Bar = 5 µm.</p

The Effect of Lat-A on actin and endocytosis.

<p>(A) Lat-A was added at the levels indicated for 15 minutes, before cells were fixed and labelled with rhodamine phalloidin to visualize F-actin. Bar = 5 µm. (B) The fluid phase dye Lucifer yellow was added to cells in the presence of 0, 25 or 400 µM Lat-A. Uptake of Lucifer yellow was assessed after 90 minutes. Bar = 5 µm. (C) Cells were treated with Lat-A 25 µM or DMSO (control) for 20 minutes before incubating with FM4-64. The localization of the dye was categorized as plasma membrane (PM), endosomal (End) or vacuolar (vac). Shown is the mean±Std Dev of 3 experiments. An unpaired students t-test indicates that there is a significant increase in endosomal staining in the treated cells p<0.0001. Examples of cells stained with FM4-64 in the absence or presence of Lat-A are shown. Bar = 5 µm (D) Strains expressing integrated GFP-Snc1 were imaged in the presence or absence of 25 µM Lat-A. Green arrowheads indicate internalising or internalized material. Red arrowheads show puncta of GFP-Snc1 at the plasma membrane. Bar = 5 µm. (E) Wild type cells expressing Sla1-GFP were grown to mid-log phase, half the sample was treated with Lat-A for 20 minutes. Time lapse movies were recorded over 90 seconds and kymographs generated.</p

The Effect of Gene Deletions on Bulk Endocytosis in the presence and absence of 25 µM Lat-A.

<p>Different endocytic null mutant strains were grown to mid log phase and incubated with bulk endocytic markers in the absence of presence of 25 µM Lat-A. (A) FM4-64 uptake was assessed after 20 minutes incubation and categorized as being plasma membrane, endosomal or vacuolar. Error bars – std deviation. (B) Analysis of the status of LY puncta following incubation in the absence or presence of Lat-A. Categories determined (i) in the plane of the membrane, (ii) invaginated or (iii) successfully undergone scission. Number of vesicles counted ≥50 in ≥10 cells. Error is std deviation.</p

The Effect of Abp1 mutations on Bulk Endocytosis.

<p>(A) Schematic of full length Abp1 and the different mutants analysed in this study. (B) Wild type, <i>abp1</i>Δ and <i>abp1Δ</i> cells expressing wild type <i>ABP1</i> 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.</p

Mutation of key lysine residues in the Insert B region of the yeast dynamin Vps1 disrupts lipid binding and causes defects in endocytosis.

The yeast dynamin-like protein Vps1 has roles at multiple stages of membrane trafficking including Golgi to vacuole transport, endosomal recycling, endocytosis and in peroxisomal fission. While the majority of the Vps1 amino acid sequence shows a high level of identity with the classical mammalian dynamins, it does not contain a pleckstrin homology domain (PH domain). The Dyn1 PH domain has been shown to bind to lipids with a preference for PI(4,5)P2 and it is considered central to the function of Dyn1 in endocytosis. The lack of a PH domain in Vps1 has raised questions as to whether the protein can function directly in membrane fusion or fission events. Here we demonstrate that the region Insert B, located in a position equivalent to the dynamin PH domain, is able to bind directly to lipids and that mutation of three lysine residues reduces its capacity to interact with lipids, and in particular with PI(4,5)P2. The Vps1 KKK-AAA mutant shows more diffuse staining but does still show some localization to compartments adjacent to vacuoles and to endocytic sites suggesting that other factors are also involved in its recruitment. This mutant selectively blocks endocytosis, but is functional in other processes tested. While mutant Vps1 can localise to endocytic sites, the mutation results in a significant increase in the lifetime of the endocytic reporter Sla2 and a high proportion of defective scission events. Together our data indicate that the lipid binding capacity of the Insert B region of Vps1 contributes to the ability of the protein to associate with membranes and that its capacity to interact with PI(4,5)P2 is important in facilitating endocytic scission