Autophagy
ISSN: 1554-8627 (Print) 1554-8635 (Online) Journal homepage: http://www.tandfonline.com/loi/kaup20
A selective autophagy pathway takes an unconventional route Xiao-Man Liu & Li-Lin Du To cite this article: Xiao-Man Liu & Li-Lin Du (2015): A selective autophagy pathway takes an unconventional route, Autophagy, DOI: 10.1080/15548627.2015.1110669 To link to this article: http://dx.doi.org/10.1080/15548627.2015.1110669
Accepted author version posted online: 13 Nov 2015.
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Date: 16 November 2015, At: 00:46
A selective autophagy pathway takes an unconventional route Xiao-Man Liu and Li-Lin Du
National Institute of Biological Sciences, Beijing 102206, China.
Downloaded by [Monash University Library] at 00:46 16 November 2015
Correspondence should be addressed to Li-Lin Du ([email protected])
Keywords
autophagy, Schizosaccharomyces pombe, Nbr1, NVT pathway, ESCRT, multivesicular body, endosomal microautophagy
Abstract
Selective autophagy transports specific cytoplasmic materials into lysosomes/vacuoles. In the case of macroautophagy the selectivity is mediated by receptors, which usually link the cargos to the machinery that sequesters them into the forming autophagosome. In our recent work, we found that fission yeast Nbr1, a homolog of the mammalian macroautophagy receptor NBR1, acts together with an unconventional autophagy-associated cargo sequestration apparatus, the endosomal sorting complexes required for transport (ESCRTs), to deliver 2 hydrolytic enzymes from the cytosol to the vacuole lumen. In this pathway, which we term the Nbr1-mediated vacuolar targeting (NVT) pathway, soluble cargos transit through the multi-vesicular body (MVB), rather than the autophagosome, on their way to the vacuole. Our findings
1
reveal a novel mode of action of macroautophagy receptors and broaden our
Downloaded by [Monash University Library] at 00:46 16 November 2015
understanding of ESCRT-mediated autophagy.
2
The lysosome/vacuole is a major compartment for cellular degradation. Cytoplasmic materials are delivered into the lysosome/vacuole through various types of autophagy. The best-understood autophagic mechanism is macroautophagy. During this process, autophagic cargos are engulfed by a double-membraned structure called the phagophore, which expands and matures into an autophagosome; subsequent fusion
Downloaded by [Monash University Library] at 00:46 16 November 2015
with the lysosome/vacuole delivers the cargo into the organelle lumen for degradation. Besides the nonselective macroautophagy that occurs during starvation, certain types of cargos including protein aggregates, organelles, and pathogens can be specifically recognized and removed through selective macroautophagy.
Receptor proteins play an essential role in selective macroautophagy. In mammals, macroautophagy receptors such as SQSTM1/p62, NBR1, CALCOCO2/NDP52, and OPTN/optineurin can simultaneously bind ubiquitin on the cargos and the LC3/Atg8 family proteins on the phagophore membrane. Mammalian NBR1 takes part in selective macroautophagy of aggregated proteins, peroxisomes and midbodies. In the budding yeast Saccharomyces cerevisiae, a number of soluble hydrolases including Ape1, Ams1 and Ape4 are delivered into the vacuole by the cytoplasm-to-vacuole targeting (Cvt) pathway. The Cvt pathway relies on the receptor protein Atg19, which like mammalian autophagy receptors can bind both its cargos and Atg proteins. The Cvt pathway may be evolutionarily related to mammalian selective macroautophagy because it has been suggested that Atg19 is a remote and cryptic homolog of mammalian NBR1.
3
We have been using the fission yeast Schizosaccharomyces pombe as a model to study autophagy. To our interest, in the fission yeast genome, there is a previously uncharacterized gene (SPBP35G2.11c) that encodes a clear-cut homolog of mammalian NBR1. It contains three ZZ-type zinc fingers and a FW domain, which are 2 types of domains also present in NBR1. We named this fission yeast protein Nbr1 and
Downloaded by [Monash University Library] at 00:46 16 November 2015
investigated whether it has autophagy-related functions.
Our experiments revealed that Nbr1 interacts with 2 M1 family aminopeptidases, Lap2 and Ape2, and facilitates their transport from the cytosol into the vacuole. The ZZ fingers of Nbr1 are necessary and sufficient for its interactions with Lap2 and Ape2, while both the ZZ fingers and the FW domain are required for the vacuolar targeting function. Similar to the way S. cerevisiae precursor Ape1 acts in the Cvt pathway, Lap2 and Ape2 are not only cargos but also essential components in this vacuolar targeting process. The extreme N-terminal region of Ape2 is involved in its interaction with Nbr1. This is reminiscent of how the budding yeast precursor Ape1 interacts with Atg19. We named this fission yeast pathway the Nbr1-mediated vacuolar targeting (NVT) pathway. Lap2 and Ape2 remain intact after entering the vacuole and probably function as resident vacuolar hydrolases. The similar roles of Nbr1 and Atg19 in the biosynthetic delivery of vacuolar hydrolases and the strong sequence homology between Nbr1 and mammalian NBR1 solidify the evolutionary connection between yeast vacuolar targeting pathways and mammalian selective autophagy.
4
We predicted that, as with the Cvt pathway, the NVT pathway should require the macroautophagy machinery. However, to our great surprise, this prediction turned out to be wrong. We found that the NVT pathway does not require core Atg proteins. Thus, the NVT pathway must employ a cargo sequestration mechanism different from that of the Cvt pathway. Without a concrete hypothesis on what sequestration mechanism may
Downloaded by [Monash University Library] at 00:46 16 November 2015
be involved, we resorted to an unbiased genetic screen, which led to the identification of a mutation in the sst2 gene that abolishes the vacuolar localization of Lap2 and Nbr1. Sst2 is a homolog of mammalian STAMBP/AMSH and functions with the ESCRTs in the MVB pathway that delivers membrane proteins into the vacuole via the MVB. To determine whether the ESCRT proteins participate in the NVT pathway, we analyzed mutants lacking sst4/vps27, sst6/vps23, vps36, or vps20, which encode components of ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III complexes, respectively, and found that they are all defective in the NVT pathway.
Membrane protein cargos of the MVB pathway are sorted into the intralumenal vesicles of the MVB. Thus, we hypothesized that ESCRTs may sequester the NVT pathway cargos at the MVB. Consistent with this idea, we found that NVT components colocalize with ESCRTs at MVBs. In the MVB pathway, ESCRTs recognize their cargos through binding to the ubiquitin molecules attached to the membrane proteins. Similarly, we found that ubiquitination of Nbr1, and/or its associated proteins, plays an important role in the NVT pathway.
5
The discovery of the NVT pathway raises some interesting possibilities. ESCRTs act in a mammalian autophagy pathway termed endosomal microautophagy (eMI). In this pathway, cytosolic proteins are recognized by HSPA8/Hsc70 and sequestered into the intralumenal vesicles of the MVB in an ESCRT-dependent manner. The NVT pathway is topologically similar to endosomal microautophagy. Thus, it will be
Downloaded by [Monash University Library] at 00:46 16 November 2015
interesting to investigate whether conventional macroautophagy receptors are involved in mammalian microautophagy. It will also be interesting to know whether NBR1 family proteins in higher eukaryotes transport NVT- and Cvt-types of hydrolase cargos. There are also intriguing questions that can be further addressed in the fission yeast. Besides ubiquitination, are there other signals needed for the NVT complex to be recognized by ESCRTs? Apart from the NVT complex, do ESCRTs sort other kinds of soluble cargos into the MVB? We hope that our future investigation will provide answers to these questions.
6
ISSN: 1554-8627 (Print) 1554-8635 (Online) Journal homepage: http://www.tandfonline.com/loi/kaup20
A selective autophagy pathway takes an unconventional route Xiao-Man Liu & Li-Lin Du To cite this article: Xiao-Man Liu & Li-Lin Du (2015): A selective autophagy pathway takes an unconventional route, Autophagy, DOI: 10.1080/15548627.2015.1110669 To link to this article: http://dx.doi.org/10.1080/15548627.2015.1110669
Accepted author version posted online: 13 Nov 2015.
Submit your article to this journal
Article views: 9
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kaup20 Download by: [Monash University Library]
Date: 16 November 2015, At: 00:46
A selective autophagy pathway takes an unconventional route Xiao-Man Liu and Li-Lin Du
National Institute of Biological Sciences, Beijing 102206, China.
Downloaded by [Monash University Library] at 00:46 16 November 2015
Correspondence should be addressed to Li-Lin Du ([email protected])
Keywords
autophagy, Schizosaccharomyces pombe, Nbr1, NVT pathway, ESCRT, multivesicular body, endosomal microautophagy
Abstract
Selective autophagy transports specific cytoplasmic materials into lysosomes/vacuoles. In the case of macroautophagy the selectivity is mediated by receptors, which usually link the cargos to the machinery that sequesters them into the forming autophagosome. In our recent work, we found that fission yeast Nbr1, a homolog of the mammalian macroautophagy receptor NBR1, acts together with an unconventional autophagy-associated cargo sequestration apparatus, the endosomal sorting complexes required for transport (ESCRTs), to deliver 2 hydrolytic enzymes from the cytosol to the vacuole lumen. In this pathway, which we term the Nbr1-mediated vacuolar targeting (NVT) pathway, soluble cargos transit through the multi-vesicular body (MVB), rather than the autophagosome, on their way to the vacuole. Our findings
1
reveal a novel mode of action of macroautophagy receptors and broaden our
Downloaded by [Monash University Library] at 00:46 16 November 2015
understanding of ESCRT-mediated autophagy.
2
The lysosome/vacuole is a major compartment for cellular degradation. Cytoplasmic materials are delivered into the lysosome/vacuole through various types of autophagy. The best-understood autophagic mechanism is macroautophagy. During this process, autophagic cargos are engulfed by a double-membraned structure called the phagophore, which expands and matures into an autophagosome; subsequent fusion
Downloaded by [Monash University Library] at 00:46 16 November 2015
with the lysosome/vacuole delivers the cargo into the organelle lumen for degradation. Besides the nonselective macroautophagy that occurs during starvation, certain types of cargos including protein aggregates, organelles, and pathogens can be specifically recognized and removed through selective macroautophagy.
Receptor proteins play an essential role in selective macroautophagy. In mammals, macroautophagy receptors such as SQSTM1/p62, NBR1, CALCOCO2/NDP52, and OPTN/optineurin can simultaneously bind ubiquitin on the cargos and the LC3/Atg8 family proteins on the phagophore membrane. Mammalian NBR1 takes part in selective macroautophagy of aggregated proteins, peroxisomes and midbodies. In the budding yeast Saccharomyces cerevisiae, a number of soluble hydrolases including Ape1, Ams1 and Ape4 are delivered into the vacuole by the cytoplasm-to-vacuole targeting (Cvt) pathway. The Cvt pathway relies on the receptor protein Atg19, which like mammalian autophagy receptors can bind both its cargos and Atg proteins. The Cvt pathway may be evolutionarily related to mammalian selective macroautophagy because it has been suggested that Atg19 is a remote and cryptic homolog of mammalian NBR1.
3
We have been using the fission yeast Schizosaccharomyces pombe as a model to study autophagy. To our interest, in the fission yeast genome, there is a previously uncharacterized gene (SPBP35G2.11c) that encodes a clear-cut homolog of mammalian NBR1. It contains three ZZ-type zinc fingers and a FW domain, which are 2 types of domains also present in NBR1. We named this fission yeast protein Nbr1 and
Downloaded by [Monash University Library] at 00:46 16 November 2015
investigated whether it has autophagy-related functions.
Our experiments revealed that Nbr1 interacts with 2 M1 family aminopeptidases, Lap2 and Ape2, and facilitates their transport from the cytosol into the vacuole. The ZZ fingers of Nbr1 are necessary and sufficient for its interactions with Lap2 and Ape2, while both the ZZ fingers and the FW domain are required for the vacuolar targeting function. Similar to the way S. cerevisiae precursor Ape1 acts in the Cvt pathway, Lap2 and Ape2 are not only cargos but also essential components in this vacuolar targeting process. The extreme N-terminal region of Ape2 is involved in its interaction with Nbr1. This is reminiscent of how the budding yeast precursor Ape1 interacts with Atg19. We named this fission yeast pathway the Nbr1-mediated vacuolar targeting (NVT) pathway. Lap2 and Ape2 remain intact after entering the vacuole and probably function as resident vacuolar hydrolases. The similar roles of Nbr1 and Atg19 in the biosynthetic delivery of vacuolar hydrolases and the strong sequence homology between Nbr1 and mammalian NBR1 solidify the evolutionary connection between yeast vacuolar targeting pathways and mammalian selective autophagy.
4
We predicted that, as with the Cvt pathway, the NVT pathway should require the macroautophagy machinery. However, to our great surprise, this prediction turned out to be wrong. We found that the NVT pathway does not require core Atg proteins. Thus, the NVT pathway must employ a cargo sequestration mechanism different from that of the Cvt pathway. Without a concrete hypothesis on what sequestration mechanism may
Downloaded by [Monash University Library] at 00:46 16 November 2015
be involved, we resorted to an unbiased genetic screen, which led to the identification of a mutation in the sst2 gene that abolishes the vacuolar localization of Lap2 and Nbr1. Sst2 is a homolog of mammalian STAMBP/AMSH and functions with the ESCRTs in the MVB pathway that delivers membrane proteins into the vacuole via the MVB. To determine whether the ESCRT proteins participate in the NVT pathway, we analyzed mutants lacking sst4/vps27, sst6/vps23, vps36, or vps20, which encode components of ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III complexes, respectively, and found that they are all defective in the NVT pathway.
Membrane protein cargos of the MVB pathway are sorted into the intralumenal vesicles of the MVB. Thus, we hypothesized that ESCRTs may sequester the NVT pathway cargos at the MVB. Consistent with this idea, we found that NVT components colocalize with ESCRTs at MVBs. In the MVB pathway, ESCRTs recognize their cargos through binding to the ubiquitin molecules attached to the membrane proteins. Similarly, we found that ubiquitination of Nbr1, and/or its associated proteins, plays an important role in the NVT pathway.
5
The discovery of the NVT pathway raises some interesting possibilities. ESCRTs act in a mammalian autophagy pathway termed endosomal microautophagy (eMI). In this pathway, cytosolic proteins are recognized by HSPA8/Hsc70 and sequestered into the intralumenal vesicles of the MVB in an ESCRT-dependent manner. The NVT pathway is topologically similar to endosomal microautophagy. Thus, it will be
Downloaded by [Monash University Library] at 00:46 16 November 2015
interesting to investigate whether conventional macroautophagy receptors are involved in mammalian microautophagy. It will also be interesting to know whether NBR1 family proteins in higher eukaryotes transport NVT- and Cvt-types of hydrolase cargos. There are also intriguing questions that can be further addressed in the fission yeast. Besides ubiquitination, are there other signals needed for the NVT complex to be recognized by ESCRTs? Apart from the NVT complex, do ESCRTs sort other kinds of soluble cargos into the MVB? We hope that our future investigation will provide answers to these questions.
6
