scholarly journals A CRISPR screening approach for identifying novel autophagy-related factors and cytoplasm-to-lysosome trafficking routes

2017 ◽  
Author(s):  
Christopher J Shoemaker ◽  
Tina Q Huang ◽  
Nicholas R Weir ◽  
Nicole Polyakov ◽  
Vladimir Denic
Keyword(s):  
Membrane Vesicles ◽  
Genetic Interactions ◽  
Endoplasmic Reticulum Membrane ◽  
Autophagosome Formation ◽  
Related Factors ◽  
Double Membrane ◽  
Receptor Proteins ◽  
Selective Autophagy ◽  
Genetic Fingerprint ◽  
Genome Wide

SummarySelective autophagy comprises cytoplasm-to-lysosome trafficking routes that transport cargos using double-membrane vesicles (autophagosomes). Cargos are detected by receptor proteins, which typically also bind to lipid-conjugated LC3 proteins on autophagosome membranes. We dissected lysosomal delivery of four SQSTM1-like receptors by genome-wide CRISPR screening looking for novel autophagy-related (ATG) factors and trafficking routes. We uncovered new mammalian ATG factors including TMEM41B, an endoplasmic reticulum membrane protein required for autophagosome membrane expansion and/or closure. Furthermore, we found that certain receptors remain robustly targeted to the lysosome even in the absence of ATG7 or other LC3 conjugation factors. Lastly, we identified a unique genetic fingerprint behind receptor flux in ATG7KO cells, which includes factors implicated in nucleating autophagosome formation and vesicle trafficking factors. Our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.

scholarly journals Pex3 confines pexophagy receptor activity of Atg36 to peroxisomes by regulating Hrr25-mediated phosphorylation and proteasomal degradation

2020 ◽  
Vol 295 (48) ◽  
pp. 16292-16298
Author(s):  
Sota Meguro ◽  
Xizhen Zhuang ◽  
Hiromi Kirisako ◽  
Hitoshi Nakatogawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Recombinant Proteins ◽  
Posttranslational Modifications ◽  
Membrane Vesicles ◽  
Proteasomal Degradation ◽  
Regulatory Mechanisms ◽  
Autophagosome Formation ◽  
Peroxisomal Membrane ◽  
Double Membrane ◽  
Selective Autophagy

In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36–Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36–Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.

scholarly journals Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum

2008 ◽  
Vol 182 (4) ◽  
pp. 685-701 ◽  
Author(s):  
Elizabeth L. Axe ◽  
Simon A. Walker ◽  
Maria Manifava ◽  
Priya Chandra ◽  
H. Llewelyn Roderick ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Dynamic Equilibrium ◽  
Membrane Vesicles ◽  
Amino Acid Starvation ◽  
Autophagosome Formation ◽  
Double Membrane ◽  
Dynamic Relationship ◽  
Golgi Membranes ◽  
Membrane Compartments ◽  
Phosphatidylinositol 3

Autophagy is the engulfment of cytosol and organelles by double-membrane vesicles termed autophagosomes. Autophagosome formation is known to require phosphatidylinositol 3-phosphate (PI(3)P) and occurs near the endoplasmic reticulum (ER), but the exact mechanisms are unknown. We show that double FYVE domain–containing protein 1, a PI(3)P-binding protein with unusual localization on ER and Golgi membranes, translocates in response to amino acid starvation to a punctate compartment partially colocalized with autophagosomal proteins. Translocation is dependent on Vps34 and beclin function. Other PI(3)P-binding probes targeted to the ER show the same starvation-induced translocation that is dependent on PI(3)P formation and recognition. Live imaging experiments show that this punctate compartment forms near Vps34-containing vesicles, is in dynamic equilibrium with the ER, and provides a membrane platform for accumulation of autophagosomal proteins, expansion of autophagosomal membranes, and emergence of fully formed autophagosomes. This PI(3)P-enriched compartment may be involved in autophagosome biogenesis. Its dynamic relationship with the ER is consistent with the idea that the ER may provide important components for autophagosome formation.

scholarly journals Atg27 Is Required for Autophagy-dependent Cycling of Atg9

2007 ◽  
Vol 18 (2) ◽  
pp. 581-593 ◽  
Author(s):  
Wei-Lien Yen ◽  
Julie E. Legakis ◽  
Usha Nair ◽  
Daniel J. Klionsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Golgi Complex ◽  
Transmembrane Protein ◽  
Eukaryotic Cells ◽  
Vesicle Formation ◽  
Catabolic Pathway ◽  
Autophagosome Formation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Double Membrane ◽  
Selective Autophagy

Autophagy is a catabolic pathway for the degradation of cytosolic proteins or organelles and is conserved among all eukaryotic cells. The hallmark of autophagy is the formation of double-membrane cytosolic vesicles, termed autophagosomes, which sequester cytoplasm; however, the mechanism of vesicle formation and the membrane source remain unclear. In the yeast Saccharomyces cerevisiae, selective autophagy mediates the delivery of specific cargos to the vacuole, the analog of the mammalian lysosome. The transmembrane protein Atg9 cycles between the mitochondria and the pre-autophagosomal structure, which is the site of autophagosome biogenesis. Atg9 is thought to mediate the delivery of membrane to the forming autophagosome. Here, we characterize a second transmembrane protein Atg27 that is required for specific autophagy in yeast. Atg27 is required for Atg9 cycling and shuttles between the pre-autophagosomal structure, mitochondria, and the Golgi complex. These data support a hypothesis that multiple membrane sources supply the lipids needed for autophagosome formation.

scholarly journals Atg39 links and deforms the outer and inner nuclear membranes in selective autophagy of the nucleus

2021 ◽  
Author(s):  
Keisuke Mochida ◽  
Toshifumi Otani ◽  
Yuto Katsumata ◽  
Hiromi Kirisako ◽  
Chika Kakuta ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Membrane ◽  
Transmembrane Domain ◽  
Membrane Vesicles ◽  
Space Region ◽  
Double Membrane ◽  
Selective Autophagy ◽  
Nuclear Membranes ◽  
Amphipathic Helices ◽  
Perinuclear Space

In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane (ONM) via its transmembrane domain and also associated with the inner nuclear membrane (INM) via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that overaccumulation of Atg39 causes the NE to protrude towards the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 assembly in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.

scholarly journals TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes

2012 ◽  
Vol 197 (5) ◽  
pp. 659-675 ◽  
Author(s):  
Andrea Longatti ◽  
Christopher A. Lamb ◽  
Minoo Razi ◽  
Shin-ichiro Yoshimura ◽  
Francis A. Barr ◽  
...  
Keyword(s):  
Amino Acid ◽  
Golgi Complex ◽  
Membrane Vesicles ◽  
Degradation Process ◽  
Amino Acid Starvation ◽  
Negative Regulators ◽  
Autophagosome Formation ◽  
Double Membrane ◽  
Recycling Endosomes ◽  
Guanosine Triphosphatase

Autophagy is a bulk degradation process characterized by the formation of double membrane vesicles called autophagosomes. The exact molecular mechanism of autophagosome formation and the origin of the autophagosomal membrane remain unclear. We screened 38 human Tre-2/Bub2/Cdc16 domain–containing Rab guanosine triphosphatase–activating proteins (GAPs) and identified 11 negative regulators of starvation-induced autophagy. One of these putative RabGAPs, TBC1D14, colocalizes and interacts with the autophagy kinase ULK1. Overexpressed TBC1D14 tubulates ULK1-positive recycling endosomes (REs), impairing their function and inhibiting autophagosome formation. TBC1D14 binds activated Rab11 but is not a GAP for Rab11, and loss of Rab11 prevents TBC1D14-induced tubulation of REs. Furthermore, Rab11 is required for autophagosome formation. ULK1 and Atg9 are found on Rab11- and transferrin (Tfn) receptor (TfnR)–positive recycling endosomes. Amino acid starvation causes TBC1D14 to relocalize from REs to the Golgi complex, whereas TfnR and Tfn localize to forming autophagosomes, which are ULK1 and LC3 positive. Thus, TBC1D14- and Rab11-dependent vesicular transport from REs contributes to and regulates starvation-induced autophagy.

scholarly journals The Actin Cytoskeleton Is Required for Selective Types of Autophagy, but Not Nonspecific Autophagy, in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 16 (12) ◽  
pp. 5843-5856 ◽  
Author(s):  
Fulvio Reggiori ◽  
Iryna Monastyrska ◽  
Takahiro Shintani ◽  
Daniel J. Klionsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Eukaryotic Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Double Membrane ◽  
Selective Autophagy ◽  
Putative Site ◽  
Vesicle Biogenesis

Autophagy is a catabolic multitask transport route that takes place in all eukaryotic cells. During starvation, cytoplasmic components are randomly sequestered into huge double-membrane vesicles called autophagosomes and delivered into the lysosome/vacuole where they are destroyed. Cells are able to modulate autophagy in response to their needs, and under certain circumstances, cargoes such as aberrant protein aggregates, organelles and bacteria can be selectively and exclusively incorporated into autophagosomes. In the yeast Saccharomyces cerevisiae, for example, double-membrane vesicles are used to transport the Ape1 protease into the vacuole, or for the elimination of superfluous peroxisomes. In the present study we reveal that in this organism, actin plays a role in these two types of selective autophagy but not in the nonselective, bulk process. In particular, we show that precursor Ape1 is not correctly recruited to the PAS, the putative site of double-membrane vesicle biogenesis, and superfluous peroxisomes are not degraded in a conditional actin mutant. These phenomena correlate with a defect in Atg9 trafficking from the mitochondria to the PAS.

scholarly journals Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

2014 ◽  
Vol 42 (15) ◽  
pp. 9838-9853 ◽  
Author(s):  
Saeed Kaboli ◽  
Takuya Yamakawa ◽  
Keisuke Sunada ◽  
Tao Takagaki ◽  
Yu Sasano ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Interaction Network ◽  
Essential Genes ◽  
Genetic Interactions ◽  
Lethal Gene ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale

Abstract Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

scholarly journals A genome-wide map of human genetic interactions inferred from radiation hybrid genotypes

2010 ◽  
Vol 20 (8) ◽  
pp. 1122-1132 ◽  
Author(s):  
A. Lin ◽  
R. T. Wang ◽  
S. Ahn ◽  
C. C. Park ◽  
D. J. Smith
Keyword(s):  
Radiation Hybrid ◽  
Genetic Interactions ◽  
Genome Wide ◽  
A Genome

Hepatic bile flow

1984 ◽  
Vol 64 (4) ◽  
pp. 1055-1102 ◽  
Author(s):  
R. C. Strange
Keyword(s):  
Bile Salt ◽  
Bile Salts ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Critical Micellar Concentration ◽  
Canalicular Membrane ◽  
Receptor Proteins ◽  
Subcellular Organelles ◽  
Golgi Bodies ◽  
Initial Event

The hepatocyte is a polar cell that can remove a variety of molecules from blood and excrete them into bile. This review is primarily concerned with the mechanism of transport of the principal anions--the bile salts--across the sinusoidal membrane, their passage through the cell, and excretion across the canalicular membrane. Clearly much of this process is poorly understood, but the study of the membrane stages should be facilitated by the ability to prepare purified sinusoidal and canalicular membrane vesicles. For example, the relative importance of albumin-binding sites as well as the putative bile salt receptor proteins can be better assessed. It seems likely that although the interaction of bile salts with receptor proteins is important, it is an initial event that puts the bile salt in the correct place for uptake to occur. The driving force for uptake is the Na+ gradient created across the basolateral membrane by the activity of the Na+-K+-ATPase. Within the cell, various modes of transport have been suggested. Several authors emphasize the importance of protein binding of bile salts, either because of their presumed ability to maintain the concentration of these anions in the hepatocyte below their critical micellar concentration or because of their putative role in transport. It is important to understand these aspects of the role of cytosolic proteins for several reasons. Knowledge of the true concentration of free bile salt within the cell should allow estimation of whether the electrochemical gradient is sufficient for bile salts to accumulate in bile without the need for active transport of molecules from the cell into the canaliculus. The compartmental model described by Strange et al. (153) offers one theoretical way of determining the concentration of free bile salt, although the problems inherent in studying amphipath binding to the membranes of subcellular organelles (31) require that the model be reevaluated by the hygroscopic-desorption method. The second role suggested for the cytosolic bile salt-binding proteins is as transport proteins. As discussed in section VI, I think it is unlikely that the proteins identified so far act in this way, and it is more likely that movement occurs by diffusion in free solution. It is also important to determine the possible involvement of subcellular organelles such as Golgi bodies. Little is known of their role in the transport of bile salts or indeed where bile salt micelles are formed.(ABSTRACT TRUNCATED AT 400 WORDS)

Genome-Wide Prediction of C. elegans Genetic Interactions

Science ◽  
2006 ◽  
Vol 311 (5766) ◽  
pp. 1481-1484 ◽  
Author(s):  
W. Zhong
Keyword(s):  
Genetic Interactions ◽  
C Elegans ◽  
Genome Wide
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