Mol. Cells 2017; 40(9): 643-654
Published online September 20, 2017
https://doi.org/10.14348/molcells.2017.0030
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: zoushenshen@njau.edu.cn
Autophagy is a degradation pathway in eukaryotic cells in which aging proteins and organelles are sequestered into double-membrane vesicles, termed autophagosomes, which fuse with vacuoles to hydrolyze cargo. The key step in autophagy is the formation of autophagosomes, which requires different kinds of vesicles, including COPII vesicles and Atg9-containing vesicles, to transport lipid double-membranes to the phagophore assembly site (PAS). In yeast, the
Keywords Atg9-containing vesicles, autophagy, golgi apparatus, Sed5, Sft1/2
Autophagy is a basic physiological process in eukaryotes. It is an essential cellular degradation process that eliminates obsolete or damaged cytoplasmic materials to maintain intra-cellular homeostasis (Feng et al., 2014). Normal levels of autophagy are required for development and stress responses, and defects in autophagy are implicated in several diseases, such as neurodegeneration and cancer (Huang and Klionsky, 2007, Mizushima et al., 2008, Ravikumar et al., 2010, Yang and Klionsky, 2010). There are two forms of autophagy: selective and non-selective. Selective autophagy chooses specific cytoplasmic components for degradation (such as the cytoplasm-to-vacuole (Cvt) pathway), and non-selective autophagy is induced by stress to degrade non-specific cellular components (such as aged and nonfunctional proteins and organelles) (Lynch-Day and Klionsky, 2010). Both types of autophagy begin with the formation of the phagophore assembly site (PAS). Most Atg (autophagy-related genes) proteins, which are recruited to the PAS, are involved in the formation and extension of the phagophore. Subsequently, phagophore lead to complete double-membrane spherical structures called autophagosome. Finally, autophagosomes, which enclose cellular components, fuse with vacuoles, and cargo is released into the vacuoles and hydrolyzed (Nakatogawa et al., 2009, Reggiori and Klionsky, 2013).
Double-membrane autophagosomes are the most typical feature of autophagy. Elongation and establishment of the autophagosome requires a protein called Atg8, an ubiquitin-like protein (Klionsky et al., 2007; Nakatogawa et al., 2007). The E1 enzyme Atg7, the E2-like enzyme Atg3 and the E3 ligase Atg5-Atg12 form a complex that conjugates the Ubl protein Atg8 to phosphatidylethanolamine (PE) (Nakatogawa et al., 2007). Atg8-PE is inserted into the double-membrane of the autophagosome after it is generated (Geng and Klionsky, 2008). The autophagosome then fuses with the vacuole, and the cargo inside the inner membrane as well as Atg8 are released into the vacuole. Finally, they are degraded by a hydrolase in the vacuole (Kirisako et al., 2000).
Atg9 is a transmembrane protein in yeast that is essential for autophagosome formation (Noda et al., 2000; Young et al., 2006). Previous studies showed that Atg9 normally cycles between the PAS and intracellular organelles (such as mitochondria and Golgi apparatus), where an Atg9-containing vesicle is generated (Noda et al., 2000; Mari et al., 2010). Atg9-containing vesicles transport lipid double-membranes, which are necessary for autophagosome formation (Mari et al., 2010). The formation of the Atg9-containing vesicle on the Golgi apparatus and its delivery to the PAS depend on Atg23 and Atg27 (Backues et al., 2015; Yamamoto et al., 2012).
Similar to Atg9-containing vesicles, COPII vesicles are believed to be involved in transporting the lipid double-membranes required for autophagosome formation (Davis and Ferro-Novick, 2015; Graef et al., 2013; Lemus et al., 2016; Tan et al., 2013). COPII vesicles transport cargo from the ER (Endoplasmic reticulum) to the Golgi apparatus in the secretory pathway. Recently, studies showed that many COPII components and regulators, such as the GTPase Ypt1, the inner COPII coat protein Sec23/Sec24 and the serine/threonine kinase Hrr25, participate in autophagy regulation (Davis and Ferro-Novick, 2015; Lynch-Day et al., 2010; Tan et al., 2013; Tanaka et al., 2014; Wang et al., 2013; 2015). As a COP II regulator, Sed5, a syntaxin family member, is a t-SNARE protein that forms a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex with Sec22, Bet1, and Bos1 to mediate the fusion of COPII vesicles with the Golgi apparatus (Hardwick and Pelham, 1992; Nichols and Pelham, 1998). Furthermore, Sed5 mediates intra-Golgi and endosome-Golgi transport with Sft1, Ykt6, Gos1 and Vti1 to form other SNARE complexes (Nichols and Pelham, 1998; Sogaard et al., 1994). In yeast cells, Sed5 also affects the anterograde transport of Atg9 from mitochondria to the PAS (Reggiori and Klionsky, 2006). Here, we explicitly showed that Sed5 is required for both the Cvt pathway and starvation-induced autophagy under both permissive temperatures and high-temperature stress. Moreover, we found that Sed5 mediated the transport of Atg8 from the Golgi apparatus to the PAS and is involved in the recruitment of Atg23 and Atg27, components of the Atg9-containing vesicle, to the Golgi apparatus. Finally, we found that the autophagy defect in Sed5 mutants can be suppressed by Sft1 or Sft2, which are involved in intra-Golgi transport. Therefore, our data suggest that Sed5 is involved in the transport of Atg8 from the Golgi apparatus to the PAS and mediates this process through Sft1 or Sft2.
The yeast strains and plasmids used in this study are listed in
To clone
For live-cell fluorescence microscopy, yeast cells were grown at 26°C in rich (YPD) or synthetic minimal (when a plasmid was used) media until they reached the log phase, at which point they were either grown at 26°C or moved to a restrictive temperature (34°C) for 1.5 h. If the cells were subjected to nitrogen starvation, they were washed, transferred to SD-N medium and incubated at 26°C for 2 h. Alternatively, they were pretreated for 30 min at a restrictive temperature (34°C), washed and transferred to SD-N medium, and then incubated at a restrictive temperature (34°C) for 2 more hours. If necessary, FM4-64 was added to a final concentration of 1.6 μM to stain the vacuole during the final hour of incubation. Cells prepared on slides were examined with a Nikon Eclipse Ti inverted research microscope (Tokyo, Japan). More than five fields were collected for each sample. Each experiment was repeated at least twice from independent colonies, and representative pictures are shown.
Immunoblotting was performed as previously described (Liang et al., 2007) and repeated at least twice. Blots were immunoblotted with mouse anti-GFP (Santa Cruz), rabbit anti-Ape1 (a gift from Y. Ohsumi), or rabbit anti-pGK1 (Ac-ris) antibodies, and ECL (Millipore) was used as the substrate.
To monitor autophagosome biogenesis, a protease protection assay was performed as previously described (Nair et al., 2011; Zou et al., 2015).
The Pho8Δ60 assay quantifying the level of autophagy was modified from that previously described (Noda and Klionsky, 2008).
The SNARE proteins Bet1, Bos1, Sed5, and Sec22 are required for the membrane fusion of COPII vesicles with the Golgi apparatus (Barlowe et al., 1994; Lian and Ferro-Novick, 1993). One of the four SNARE proteins, Sed5, was found to affect the anterograde transport of Atg9 (Reggiori and Klionsky, 2006). To clarify whether Sed5 is required for autophagy, we used the GFP-Atg8 processing assay to monitor autophagy. The
To extend our analysis, we used the Ape1 processing assay to elucidate the role of Sed5 in the Cvt pathway. As expected, wild-type cells primarily contained the mature form of Ape1 (mApe1) at 26°C and 34°C (Fig. 1B). In contrast,
In
Because the
Sed5 is a component of COPII vesicles and the Golgi apparatus fusion machinery (Barlowe et al., 1994). COPII vesicles also contain the Rab GTPase Ypt1 and the v-SNARE protein Bos1, which are required for membrane fusion (Lian and Ferro-Novick, 1993; Segev et al., 1988), and these two proteins have been shown to be involved in autophagy (Lynch-Day et al., 2010; Tan et al., 2013). Therefore, we examined whether these components of COPII vesicle mutants (
Atg8 transport to the PAS depends on Atg9, a transmembrane protein that is localized to several cytoplasmic sites, including the Golgi apparatus and mitochondria (He and Klionsky, 2007). Previous studies found that the anterograde transport of Atg9 from mitochondria to the PAS is blocked in
Blocking Atg9 anterograde transport leads to defective autophagosome formation (Yamamoto et al., 2012). We used the GFP-Atg8 protease protection assay to determine that autophagosome biogenesis is defective in
Normally, transporting Atg8 and Atg9 to the PAS requires Sed5 (Fig. 4) (Reggiori and Klionsky, 2006). Therefore, we examined whether Sed5 localized to the PAS, resulting in the mislocalization of Atg8 and Atg9. In WT cells, the PAS and phagophore were not completely indistinguishable, whereas in
During autophagy and the Cvt pathway, an ubiquitin-like conjugation system conjugates Atg8 to PE (Ichimura et al., 2000), and targeting Atg8 to the PAS requires the formation of Atg8-PE (Suzuki et al., 2007). Therefore, we explored whether multiple GFP-Atg8 dots were formed before or after Atg8-PE formation in the
Recruiting Atg8-PE to the PAS requires Atg9 (Suzuki et al., 2007), a transmembrane protein that shuttles between the PAS and other peripheral sites to provide a membrane to the PAS, which is indispensable for the formation of autophagosomes (Feng et al., 2014). Atg14 is an autophagy-specific subunit of class III PIK3 complex I that targets complex I to the PAS (Obara et al., 2006). Trs85 is a specific subunit of TRAPP III, a multimeric guanine nucleotide-exchange factor for the GTPase Ypt1, required for autophagy and the CVT pathway (Lynch-Day et al., 2010). We deleted
As Figs. 4 and 5 shown that multiple Atg8 dots in
Atg8 transport from the Golgi apparatus to the PAS also depends on Atg9-containing vesicles. The Atg23-Atg9-Atg27 complex regulates the formation of Atg9 in the Golgi apparatus, and Atg9-containing vesicles are delivered to the PAS so that autophagosomes can be formed (Backues et al., 2015; Yen et al., 2007). During this process, Atg9, Atg23 and Atg27 cycle between being localized in several cytoplasmic sites (including the Golgi apparatus and mitochondria) and the PAS (Backues et al., 2015; Reggiori et al., 2004). Because the Atg23-Atg9-Atg27 complex works upstream of Atg8 and Atg8 is trapped in the Golgi apparatus in
Atg23 trafficking cycles between organelles and the PAS, and recycling Atg23 from the PAS depends on Atg1-Atg13 (Reggiori et al., 2004). Thus, in the
Sft1 is a SNARE protein that binds Sed5 and is required for vesicle transport within the Golgi compartment (Banfield et al., 1995; Parlati et al., 2002; Wooding and Pelham, 1998). Sft2 is a non-essential membrane protein that also localizes to a late-Golgi compartment and is involved in vesicle fusion with the Golgi complex (Conchon et al., 1999; Wooding and Pelham, 1998). Both Sft1 and Sft2 exhibit genetic interactions with Sed5, as Sft1 or Sft2 overexpression could suppress the temperature sensitivity of the
Autophagy is a highly conserved physiological process in eukaryotic organisms, and autophagosome formation is a key step in this process. Autophagosomes are composed of a double lipid membrane structure, and elucidating the source of these double lipid membranes has been an important problem in the field of autophagy (Stanley et al., 2014). The present study showed that the ER, Golgi apparatus, mitochondria and endosomes act as sources of autophagosome double lipid membranes (Geng and Klionsky, 2010; Hailey et al., 2010; Mari and Reggiori, 2010; Yla-Anttila et al., 2009). COPII-coated vesicles perform transport from the ER to the Golgi apparatus or from the ER to the PAS, and most COPII vesicles protein components and regulators participate in both transport pathways (Davis and Ferro-Novick, 2015; Lemus and Goder, 2016; Wang et al., 2014). For example, the TRAPPI complex recruits Ypt1 to COPII vesicles and is involved in docking COPII to the Golgi apparatus (Cai et al., 2007). Similarly, the TRAPPIII complex also recruits Ypt1 to COPII vesicles, which helps transport COPII to the PAS (Tan et al., 2013). Sed5, a component of COPII vesicles, is involved in the fusion of COPII vesicles and the Golgi apparatus. In addition, syntaxin-5, a human homologue of Sed5, regulates the later stages of autophagy after autophagosomes are initially formed by regulating ER-to-Golgi transport (Renna et al., 2011). In this study, we clarify that Sed5 is involved in both the Cvt pathway and starvation-induced autophagy (Fig. 1), but unlike the findings of Renna et al., we found that Sed5 plays a role in the formation of autophagosomes in yeast (
Atg8 is an autophagy marker protein often used to track the autophagy process when tagged with GFP. We found that GFP-Atg8 was mislocalized at permissive and non-permissive temperatures in
Previous studies found that Sed5 is required for the anterograde transport of Atg9 from mitochondria to the PAS (Reggiori and Klionsky, 2006), but multiple GFP-Atg8 dots were not trapped in the mitochondria of
Sed5, localized in the c
Previous work showed that Sed5 participated in the anterograde transport of Atg9 from mitochondria to the PAS (Reggiori and Klionsky, 2006). Atg9, the core protein of Atg9-containing vesicles, also forms the Atg9 complex with the autophagy-related proteins Atg23 and Atg27, which are localized in the Golgi apparatus and are involved in the generation of Atg9-containing vesicles from the Golgi apparatus (Backues et al., 2015). Thus, this complex helps Atg9-containing vesicles transport cargo (such as membrane autophagosomes) to the PAS (Backues et al., 2015; Legakis et al., 2007; Yen et al., 2007). In this study, we found that Atg9 was normally localized in the Golgi apparatus in
Sft1 and Sft2 are localized in the Golgi apparatus, and these two proteins are the suppressors of
In summary, our results found that the Atg8 protein accumulated on the Golgi apparatus in s
(A) GFP-Atg8 processing was blocked in
(A) Abnormal Atg8 localization in the Cvt pathway and autophagy in
(A) GFP-Atg8 and Tom20-RFP were integrated into the chromosomes of WT and
GFP-Atg8 and RFP-Ape1 were integrated into the chromosomes of WT,
Multiple GFP-Atg8 dots disappeared in the
(A–C) WT and
(A–C) WT and
(A) Sft1 and Sft2 suppressed the defective transport of GFP-Atg8 in the
Sed5 was required for Atg23 and Atg27 localization to the Golgi apparatus, wherein the Atg9 complex (including Atg9, Atg27 and Atg23) forms and regulates Atg9-containing vesicles formation and transport to the PAS.
Mol. Cells 2017; 40(9): 643-654
Published online September 30, 2017 https://doi.org/10.14348/molcells.2017.0030
Copyright © The Korean Society for Molecular and Cellular Biology.
Shenshen Zou*, Dan Sun, and Yongheng Liang
College of Life Sciences, Key Laboratory of Agricultural Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
Correspondence to:*Correspondence: zoushenshen@njau.edu.cn
Autophagy is a degradation pathway in eukaryotic cells in which aging proteins and organelles are sequestered into double-membrane vesicles, termed autophagosomes, which fuse with vacuoles to hydrolyze cargo. The key step in autophagy is the formation of autophagosomes, which requires different kinds of vesicles, including COPII vesicles and Atg9-containing vesicles, to transport lipid double-membranes to the phagophore assembly site (PAS). In yeast, the
Keywords: Atg9-containing vesicles, autophagy, golgi apparatus, Sed5, Sft1/2
Autophagy is a basic physiological process in eukaryotes. It is an essential cellular degradation process that eliminates obsolete or damaged cytoplasmic materials to maintain intra-cellular homeostasis (Feng et al., 2014). Normal levels of autophagy are required for development and stress responses, and defects in autophagy are implicated in several diseases, such as neurodegeneration and cancer (Huang and Klionsky, 2007, Mizushima et al., 2008, Ravikumar et al., 2010, Yang and Klionsky, 2010). There are two forms of autophagy: selective and non-selective. Selective autophagy chooses specific cytoplasmic components for degradation (such as the cytoplasm-to-vacuole (Cvt) pathway), and non-selective autophagy is induced by stress to degrade non-specific cellular components (such as aged and nonfunctional proteins and organelles) (Lynch-Day and Klionsky, 2010). Both types of autophagy begin with the formation of the phagophore assembly site (PAS). Most Atg (autophagy-related genes) proteins, which are recruited to the PAS, are involved in the formation and extension of the phagophore. Subsequently, phagophore lead to complete double-membrane spherical structures called autophagosome. Finally, autophagosomes, which enclose cellular components, fuse with vacuoles, and cargo is released into the vacuoles and hydrolyzed (Nakatogawa et al., 2009, Reggiori and Klionsky, 2013).
Double-membrane autophagosomes are the most typical feature of autophagy. Elongation and establishment of the autophagosome requires a protein called Atg8, an ubiquitin-like protein (Klionsky et al., 2007; Nakatogawa et al., 2007). The E1 enzyme Atg7, the E2-like enzyme Atg3 and the E3 ligase Atg5-Atg12 form a complex that conjugates the Ubl protein Atg8 to phosphatidylethanolamine (PE) (Nakatogawa et al., 2007). Atg8-PE is inserted into the double-membrane of the autophagosome after it is generated (Geng and Klionsky, 2008). The autophagosome then fuses with the vacuole, and the cargo inside the inner membrane as well as Atg8 are released into the vacuole. Finally, they are degraded by a hydrolase in the vacuole (Kirisako et al., 2000).
Atg9 is a transmembrane protein in yeast that is essential for autophagosome formation (Noda et al., 2000; Young et al., 2006). Previous studies showed that Atg9 normally cycles between the PAS and intracellular organelles (such as mitochondria and Golgi apparatus), where an Atg9-containing vesicle is generated (Noda et al., 2000; Mari et al., 2010). Atg9-containing vesicles transport lipid double-membranes, which are necessary for autophagosome formation (Mari et al., 2010). The formation of the Atg9-containing vesicle on the Golgi apparatus and its delivery to the PAS depend on Atg23 and Atg27 (Backues et al., 2015; Yamamoto et al., 2012).
Similar to Atg9-containing vesicles, COPII vesicles are believed to be involved in transporting the lipid double-membranes required for autophagosome formation (Davis and Ferro-Novick, 2015; Graef et al., 2013; Lemus et al., 2016; Tan et al., 2013). COPII vesicles transport cargo from the ER (Endoplasmic reticulum) to the Golgi apparatus in the secretory pathway. Recently, studies showed that many COPII components and regulators, such as the GTPase Ypt1, the inner COPII coat protein Sec23/Sec24 and the serine/threonine kinase Hrr25, participate in autophagy regulation (Davis and Ferro-Novick, 2015; Lynch-Day et al., 2010; Tan et al., 2013; Tanaka et al., 2014; Wang et al., 2013; 2015). As a COP II regulator, Sed5, a syntaxin family member, is a t-SNARE protein that forms a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex with Sec22, Bet1, and Bos1 to mediate the fusion of COPII vesicles with the Golgi apparatus (Hardwick and Pelham, 1992; Nichols and Pelham, 1998). Furthermore, Sed5 mediates intra-Golgi and endosome-Golgi transport with Sft1, Ykt6, Gos1 and Vti1 to form other SNARE complexes (Nichols and Pelham, 1998; Sogaard et al., 1994). In yeast cells, Sed5 also affects the anterograde transport of Atg9 from mitochondria to the PAS (Reggiori and Klionsky, 2006). Here, we explicitly showed that Sed5 is required for both the Cvt pathway and starvation-induced autophagy under both permissive temperatures and high-temperature stress. Moreover, we found that Sed5 mediated the transport of Atg8 from the Golgi apparatus to the PAS and is involved in the recruitment of Atg23 and Atg27, components of the Atg9-containing vesicle, to the Golgi apparatus. Finally, we found that the autophagy defect in Sed5 mutants can be suppressed by Sft1 or Sft2, which are involved in intra-Golgi transport. Therefore, our data suggest that Sed5 is involved in the transport of Atg8 from the Golgi apparatus to the PAS and mediates this process through Sft1 or Sft2.
The yeast strains and plasmids used in this study are listed in
To clone
For live-cell fluorescence microscopy, yeast cells were grown at 26°C in rich (YPD) or synthetic minimal (when a plasmid was used) media until they reached the log phase, at which point they were either grown at 26°C or moved to a restrictive temperature (34°C) for 1.5 h. If the cells were subjected to nitrogen starvation, they were washed, transferred to SD-N medium and incubated at 26°C for 2 h. Alternatively, they were pretreated for 30 min at a restrictive temperature (34°C), washed and transferred to SD-N medium, and then incubated at a restrictive temperature (34°C) for 2 more hours. If necessary, FM4-64 was added to a final concentration of 1.6 μM to stain the vacuole during the final hour of incubation. Cells prepared on slides were examined with a Nikon Eclipse Ti inverted research microscope (Tokyo, Japan). More than five fields were collected for each sample. Each experiment was repeated at least twice from independent colonies, and representative pictures are shown.
Immunoblotting was performed as previously described (Liang et al., 2007) and repeated at least twice. Blots were immunoblotted with mouse anti-GFP (Santa Cruz), rabbit anti-Ape1 (a gift from Y. Ohsumi), or rabbit anti-pGK1 (Ac-ris) antibodies, and ECL (Millipore) was used as the substrate.
To monitor autophagosome biogenesis, a protease protection assay was performed as previously described (Nair et al., 2011; Zou et al., 2015).
The Pho8Δ60 assay quantifying the level of autophagy was modified from that previously described (Noda and Klionsky, 2008).
The SNARE proteins Bet1, Bos1, Sed5, and Sec22 are required for the membrane fusion of COPII vesicles with the Golgi apparatus (Barlowe et al., 1994; Lian and Ferro-Novick, 1993). One of the four SNARE proteins, Sed5, was found to affect the anterograde transport of Atg9 (Reggiori and Klionsky, 2006). To clarify whether Sed5 is required for autophagy, we used the GFP-Atg8 processing assay to monitor autophagy. The
To extend our analysis, we used the Ape1 processing assay to elucidate the role of Sed5 in the Cvt pathway. As expected, wild-type cells primarily contained the mature form of Ape1 (mApe1) at 26°C and 34°C (Fig. 1B). In contrast,
In
Because the
Sed5 is a component of COPII vesicles and the Golgi apparatus fusion machinery (Barlowe et al., 1994). COPII vesicles also contain the Rab GTPase Ypt1 and the v-SNARE protein Bos1, which are required for membrane fusion (Lian and Ferro-Novick, 1993; Segev et al., 1988), and these two proteins have been shown to be involved in autophagy (Lynch-Day et al., 2010; Tan et al., 2013). Therefore, we examined whether these components of COPII vesicle mutants (
Atg8 transport to the PAS depends on Atg9, a transmembrane protein that is localized to several cytoplasmic sites, including the Golgi apparatus and mitochondria (He and Klionsky, 2007). Previous studies found that the anterograde transport of Atg9 from mitochondria to the PAS is blocked in
Blocking Atg9 anterograde transport leads to defective autophagosome formation (Yamamoto et al., 2012). We used the GFP-Atg8 protease protection assay to determine that autophagosome biogenesis is defective in
Normally, transporting Atg8 and Atg9 to the PAS requires Sed5 (Fig. 4) (Reggiori and Klionsky, 2006). Therefore, we examined whether Sed5 localized to the PAS, resulting in the mislocalization of Atg8 and Atg9. In WT cells, the PAS and phagophore were not completely indistinguishable, whereas in
During autophagy and the Cvt pathway, an ubiquitin-like conjugation system conjugates Atg8 to PE (Ichimura et al., 2000), and targeting Atg8 to the PAS requires the formation of Atg8-PE (Suzuki et al., 2007). Therefore, we explored whether multiple GFP-Atg8 dots were formed before or after Atg8-PE formation in the
Recruiting Atg8-PE to the PAS requires Atg9 (Suzuki et al., 2007), a transmembrane protein that shuttles between the PAS and other peripheral sites to provide a membrane to the PAS, which is indispensable for the formation of autophagosomes (Feng et al., 2014). Atg14 is an autophagy-specific subunit of class III PIK3 complex I that targets complex I to the PAS (Obara et al., 2006). Trs85 is a specific subunit of TRAPP III, a multimeric guanine nucleotide-exchange factor for the GTPase Ypt1, required for autophagy and the CVT pathway (Lynch-Day et al., 2010). We deleted
As Figs. 4 and 5 shown that multiple Atg8 dots in
Atg8 transport from the Golgi apparatus to the PAS also depends on Atg9-containing vesicles. The Atg23-Atg9-Atg27 complex regulates the formation of Atg9 in the Golgi apparatus, and Atg9-containing vesicles are delivered to the PAS so that autophagosomes can be formed (Backues et al., 2015; Yen et al., 2007). During this process, Atg9, Atg23 and Atg27 cycle between being localized in several cytoplasmic sites (including the Golgi apparatus and mitochondria) and the PAS (Backues et al., 2015; Reggiori et al., 2004). Because the Atg23-Atg9-Atg27 complex works upstream of Atg8 and Atg8 is trapped in the Golgi apparatus in
Atg23 trafficking cycles between organelles and the PAS, and recycling Atg23 from the PAS depends on Atg1-Atg13 (Reggiori et al., 2004). Thus, in the
Sft1 is a SNARE protein that binds Sed5 and is required for vesicle transport within the Golgi compartment (Banfield et al., 1995; Parlati et al., 2002; Wooding and Pelham, 1998). Sft2 is a non-essential membrane protein that also localizes to a late-Golgi compartment and is involved in vesicle fusion with the Golgi complex (Conchon et al., 1999; Wooding and Pelham, 1998). Both Sft1 and Sft2 exhibit genetic interactions with Sed5, as Sft1 or Sft2 overexpression could suppress the temperature sensitivity of the
Autophagy is a highly conserved physiological process in eukaryotic organisms, and autophagosome formation is a key step in this process. Autophagosomes are composed of a double lipid membrane structure, and elucidating the source of these double lipid membranes has been an important problem in the field of autophagy (Stanley et al., 2014). The present study showed that the ER, Golgi apparatus, mitochondria and endosomes act as sources of autophagosome double lipid membranes (Geng and Klionsky, 2010; Hailey et al., 2010; Mari and Reggiori, 2010; Yla-Anttila et al., 2009). COPII-coated vesicles perform transport from the ER to the Golgi apparatus or from the ER to the PAS, and most COPII vesicles protein components and regulators participate in both transport pathways (Davis and Ferro-Novick, 2015; Lemus and Goder, 2016; Wang et al., 2014). For example, the TRAPPI complex recruits Ypt1 to COPII vesicles and is involved in docking COPII to the Golgi apparatus (Cai et al., 2007). Similarly, the TRAPPIII complex also recruits Ypt1 to COPII vesicles, which helps transport COPII to the PAS (Tan et al., 2013). Sed5, a component of COPII vesicles, is involved in the fusion of COPII vesicles and the Golgi apparatus. In addition, syntaxin-5, a human homologue of Sed5, regulates the later stages of autophagy after autophagosomes are initially formed by regulating ER-to-Golgi transport (Renna et al., 2011). In this study, we clarify that Sed5 is involved in both the Cvt pathway and starvation-induced autophagy (Fig. 1), but unlike the findings of Renna et al., we found that Sed5 plays a role in the formation of autophagosomes in yeast (
Atg8 is an autophagy marker protein often used to track the autophagy process when tagged with GFP. We found that GFP-Atg8 was mislocalized at permissive and non-permissive temperatures in
Previous studies found that Sed5 is required for the anterograde transport of Atg9 from mitochondria to the PAS (Reggiori and Klionsky, 2006), but multiple GFP-Atg8 dots were not trapped in the mitochondria of
Sed5, localized in the c
Previous work showed that Sed5 participated in the anterograde transport of Atg9 from mitochondria to the PAS (Reggiori and Klionsky, 2006). Atg9, the core protein of Atg9-containing vesicles, also forms the Atg9 complex with the autophagy-related proteins Atg23 and Atg27, which are localized in the Golgi apparatus and are involved in the generation of Atg9-containing vesicles from the Golgi apparatus (Backues et al., 2015). Thus, this complex helps Atg9-containing vesicles transport cargo (such as membrane autophagosomes) to the PAS (Backues et al., 2015; Legakis et al., 2007; Yen et al., 2007). In this study, we found that Atg9 was normally localized in the Golgi apparatus in
Sft1 and Sft2 are localized in the Golgi apparatus, and these two proteins are the suppressors of
In summary, our results found that the Atg8 protein accumulated on the Golgi apparatus in s
(A) GFP-Atg8 processing was blocked in
(A) Abnormal Atg8 localization in the Cvt pathway and autophagy in
(A) GFP-Atg8 and Tom20-RFP were integrated into the chromosomes of WT and
GFP-Atg8 and RFP-Ape1 were integrated into the chromosomes of WT,
Multiple GFP-Atg8 dots disappeared in the
(A–C) WT and
(A–C) WT and
(A) Sft1 and Sft2 suppressed the defective transport of GFP-Atg8 in the
Sed5 was required for Atg23 and Atg27 localization to the Golgi apparatus, wherein the Atg9 complex (including Atg9, Atg27 and Atg23) forms and regulates Atg9-containing vesicles formation and transport to the PAS.
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(A) GFP-Atg8 processing was blocked in
(A) Abnormal Atg8 localization in the Cvt pathway and autophagy in
(A) GFP-Atg8 and Tom20-RFP were integrated into the chromosomes of WT and
GFP-Atg8 and RFP-Ape1 were integrated into the chromosomes of WT,
Multiple GFP-Atg8 dots disappeared in the
(A–C) WT and
(A–C) WT and
(A) Sft1 and Sft2 suppressed the defective transport of GFP-Atg8 in the
Sed5 was required for Atg23 and Atg27 localization to the Golgi apparatus, wherein the Atg9 complex (including Atg9, Atg27 and Atg23) forms and regulates Atg9-containing vesicles formation and transport to the PAS.