RAB24 belongs to a family of small GTPases and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy shows an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicate that this is due to a hindrance in their clearance. Formation of autophagosomes is shown to be unaffected by RAB24-silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 is also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions.
aggregate; autophagosome; autophagy; basal; membrane; RAB24; starvation
Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation.
autophagy; electron tomography; Golgi complex; immunoEM; lysosome; mitochondrion; serial block face scanning electron microscopy; three dimensional; 3D, 3 dimensional; ATG, autophagy-related; BSA, bovine serum albumin; COPII, coat protein II; ER, endoplasmic reticulum; ET, electron tomography; GOLGA2/GM130, golgin A2; immunoEM, immuno electron microscopy; LAMP1, lysosomal-associated membrane protein 1; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MCS, membrane contact site; PBS, phosphate-buffered saline; SB-EM, serial block-face scanning electron microscopy; SEC31A, SEC31 homolog A (S. cerevisiae); TFRC, transferrin receptor; WIPI2, WD repeat domain, phosphoinositide interacting 2
The mechanisms of endosomal and lysosomal cholesterol traffic are still poorly understood. We showed previously that unesterified cholesterol accumulates in the late endosomes and lysosomes of fibroblasts deficient in both lysosome associated membrane protein-2 (LAMP-2) and LAMP-1, two abundant membrane proteins of late endosomes and lysosomes. In this study we show that in cells deficient in both LAMP-1 and LAMP-2 (LAMP−/−), low-density lipoprotein (LDL) receptor levels and LDL uptake are increased as compared to wild-type cells. However, there is a defect in esterification of both endogenous and LDL cholesterol. These results suggest that LAMP−/− cells have a defect in cholesterol transport to the site of esterification in the endoplasmic reticulum, likely due to defective export of cholesterol out of late endosomes or lysosomes. We also show that cholesterol accumulates in LAMP-2 deficient liver and that overexpression of LAMP-2 retards the lysosomal cholesterol accumulation induced by U18666A. These results point to a critical role for LAMP-2 in endosomal/lysosomal cholesterol export. Moreover, the late endosomal/lysosomal cholesterol accumulation in LAMP−/− cells was diminished by overexpression of any of the three isoforms of LAMP-2, but not by LAMP-1. The LAMP-2 luminal domain, the membrane-proximal half in particular, was necessary and sufficient for the rescue effect. Taken together, our results suggest that LAMP-2, its luminal domain in particular, plays a critical role in endosomal cholesterol transport and that this is distinct from the chaperone-mediated autophagy function of LAMP-2.
LAMP-2; LAMP-1; cholesterol; LDL; lysosome; late endosome; NPC2
autolysosome; autophagosome; chaperone-mediated autophagy; flux; LC3; lysosome; macroautophagy; phagophore; stress; vacuole
The autophagy protein BECN1/Beclin 1 is known to play a central role in autophagosome formation and maturation. The results presented here demonstrate that BECN1 interacts with the Parkinson disease-related protein PARK2. This interaction does not require PARK2 translocation to mitochondria and occurs mostly in cytosol. However, our results suggest that BECN1 is involved in PARK2 translocation to mitochondria because loss of BECN1 inhibits CCCP- or PINK1 overexpression-induced PARK2 translocation. Our results also demonstrate that the observed PARK2-BECN1 interaction is functionally important. Measurements of the level of MFN2 (mitofusin 2), a PARK2 substrate, demonstrate that depletion of BECN1 prevents PARK2 translocation-induced MFN2 ubiquitination and loss. BECN1 depletion also rescues the MFN2 loss-induced suppression of mitochondrial fusion. In sum, our results demonstrate that BECN1 interacts with PARK2 and regulates PARK2 translocation to mitochondria as well as PARK2-induced mitophagy prior to autophagosome formation.
PARK2 translocation; BECN1; mitophagy; mitochondrial dynamics; MFN2
When an autophagosome or an amphisome fuse with a lysosome, the resulting compartment is referred to as an autolysosome. Some people writing papers on the topic of autophagy use the terms “autolysosome” and “autophagolysosome” interchangeably. We contend that these words should be used to denote 2 different compartments, and that it is worthwhile maintaining this distinction—the autophagolysosome has a particular origin in the process of xenophagy that makes it distinct from an autolysosome.
amphisome; lysosome; phagophore; stress; xenophagy
The molecular mechanism of autophagy and its relationship to other lysosomal degradation pathways remain incompletely understood. Here, we identified a previously uncharacterized mammalian-specific protein, Beclin 2, which like Beclin 1, functions in autophagy and interacts with class III PI3K complex components and Bcl-2. However, Beclin 2, but not Beclin 1, functions in an additional lysosomal degradation pathway. Beclin 2 is required for ligand-induced endolysosomal degradation of several G protein-coupled receptors (GPCRs) through its interaction with GASP1. Beclin 2 homozygous knockout mice have decreased embryonic viability, and heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, and obesity and insulin resistance. Our findings identify Beclin 2 as a novel converging regulator of autophagy and GPCR turnover, and highlight the functional and mechanistic diversity of Beclin family members in autophagy, endolysosomal trafficking and metabolism.
The intramembrane protease SPPL2a cleaves the NTF of invariant chain (CD74), which is essential for normal trafficking of MHC class II–containing endosomes and thus for B cell development and function.
Regulated intramembrane proteolysis is a central cellular process involved in signal transduction and membrane protein turnover. The presenilin homologue signal-peptide-peptidase-like 2a (SPPL2a) has been implicated in the cleavage of type 2 transmembrane proteins. We show that the invariant chain (li, CD74) of the major histocompatability class II complex (MHCII) undergoes intramembrane proteolysis mediated by SPPL2a. B lymphocytes of SPPL2a−/− mice accumulate an N-terminal fragment (NTF) of CD74, which severely impairs membrane traffic within the endocytic system and leads to an altered response to B cell receptor stimulation, reduced BAFF-R surface expression, and accumulation of MHCII in transitional developmental stage T1 B cells. This results in significant loss of B cell subsets beyond the T1 stage and disrupted humoral immune responses, which can be recovered by additional ablation of CD74. Hence, we provide evidence that regulation of CD74-NTF levels by SPPL2a is indispensable for B cell development and function by maintaining trafficking and integrity of MHCII-containing endosomes, highlighting SPPL2a as a promising pharmacological target for depleting and/or modulating B cells.
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
LC3; autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
The study of autophagy is rapidly expanding, and our knowledge of the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. The vocabulary associated with autophagy has grown concomitantly. In fact, it is difficult for readers—even those who work in the field—to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors and chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, and the roles of accessory components and structures that are associated with autophagy.
autophagy; lysosome; mitophagy; pexophagy; stress; vacuole
Cdc48/p97/VCP plays a ubiquitin-independent role during autophagosome formation in S. cerevisiae.
The molecular details of the biogenesis of double-membraned autophagosomes are poorly understood. We identify the Saccharomyces cerevisiae AAA–adenosine triphosphatase Cdc48 and its substrate-recruiting cofactor Shp1/Ubx1 as novel components needed for autophagosome biogenesis. In mammals, the Cdc48 homologue p97/VCP and the Shp1 homologue p47 mediate Golgi reassembly by extracting an unknown monoubiquitinated fusion regulator from a complex. We find no requirement of ubiquitination or the proteasome system for autophagosome biogenesis but detect interaction of Shp1 with the ubiquitin-fold autophagy protein Atg8. Atg8 coupled to phosphatidylethanolamine (PE) is crucial for autophagosome elongation and, in vitro, mediates tethering and hemifusion. Interaction with Shp1 requires an FK motif within the N-terminal non–ubiquitin-like Atg8 domain. Based on our data, we speculate that autophagosome formation, in contrast to Golgi reassembly, requires a complex in which Atg8 functionally substitutes ubiquitin. This, for the first time, would give a rationale for use of the ubiquitin-like Atg8 during macroautophagy and would explain why Atg8-PE delipidation is necessary for efficient macroautophagy.
The pathogenesis of age-related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE-19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG-132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells.
autophagosome; Hsp70; lysosome; proteasome; proteolysis; retinal pigment epithelium
Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
Ubiquitously expressed micro- and millicalpain, which both require the calpain small 1 (CAPNS1) regulatory subunit for function, play important roles in numerous biological and pathological phenomena. We have previously shown that the product of GAS2, a gene specifically induced at growth arrest, is an inhibitor of millicalpain and that its overexpression sensitizes cells to apoptosis in a p53-dependent manner (Benetti, R., G. Del Sal, M. Monte, G. Paroni, C. Brancolini, and C. Schneider. 2001. EMBO J. 20:2702–2714). More recently, we have shown that calpain is also involved in nuclear factor κB activation and its relative prosurvival function in response to ceramide, in which calpain deficiency strengthens the proapoptotic effect of ceramide (Demarchi, F., C. Bertoli, P.A. Greer, and C. Schneider. 2005. Cell Death Differ. 12:512–522). Here, we further explore the involvement of calpain in the apoptotic switch and find that in calpain-deficient cells, autophagy is impaired with a resulting dramatic increase in apoptotic cell death. Immunostaining of the endogenous autophagosome marker LC3 and electron microscopy experiments demonstrate that autophagy is impaired in CAPNS1-deficient cells. Accordingly, the enhancement of lysosomal activity and long-lived protein degradation, which normally occur upon starvation, is also reduced. In CAPNS1-depleted cells, ectopic LC3 accumulates in early endosome-like vesicles that may represent a salvage pathway for protein degradation when autophagy is defective.
Mice double deficient in LAMP-1 and -2 were generated. The embryos died between embryonic days 14.5 and 16.5. An accumulation of autophagic vacuoles was detected in many tissues including endothelial cells and Schwann cells. Fibroblast cell lines derived from the double-deficient embryos accumulated autophagic vacuoles and the autophagy protein LC3II after amino acid starvation. Lysosomal vesicles were larger and more peripherally distributed and showed a lower specific density in Percoll gradients in double deficient when compared with control cells. Lysosomal enzyme activities, cathepsin D processing and mannose-6-phosphate receptor expression levels were not affected by the deficiency of both LAMPs. Surprisingly, LAMP-1 and -2 deficiencies did not affect long-lived protein degradation rates, including proteolysis due to chaperone-mediated autophagy. The LAMP-1/2 double-deficient cells and, to a lesser extent, LAMP-2 single-deficient cells showed an accumulation of unesterified cholesterol in endo/lysosomal, rab7, and NPC1 positive compartments as well as reduced amounts of lipid droplets. The cholesterol accumulation in LAMP-1/2 double-deficient cells could be rescued by overexpression of murine LAMP-2a, but not by LAMP-1, highlighting the more prominent role of LAMP-2. Taken together these findings indicate partially overlapping functions for LAMP-1 and -2 in lysosome biogenesis, autophagy, and cholesterol homeostasis.
Malignant cells often display defects in autophagy, an evolutionarily conserved pathway for degrading long-lived proteins and cytoplasmic organelles. However, as yet, there is no genetic evidence for a role of autophagy genes in tumor suppression. The beclin 1 autophagy gene is monoallelically deleted in 40–75% of cases of human sporadic breast, ovarian, and prostate cancer. Therefore, we used a targeted mutant mouse model to test the hypothesis that monoallelic deletion of beclin 1 promotes tumorigenesis. Here we show that heterozygous disruption of beclin 1 increases the frequency of spontaneous malignancies and accelerates the development of hepatitis B virus–induced premalignant lesions. Molecular analyses of tumors in beclin 1 heterozygous mice show that the remaining wild-type allele is neither mutated nor silenced. Furthermore, beclin 1 heterozygous disruption results in increased cellular proliferation and reduced autophagy in vivo. These findings demonstrate that beclin 1 is a haplo-insufficient tumor-suppressor gene and provide genetic evidence that autophagy is a novel mechanism of cell-growth control and tumor suppression. Thus, mutation of beclin 1 or other autophagy genes may contribute to the pathogenesis of human cancers.
SNARE proteins participate in recognition and fusion of membranes. A SNARE complex consisting of vti1b, syntaxin 8, syntaxin 7, and endobrevin/VAMP-8 which is required for fusion of late endosomes in vitro has been identified recently. Here, we generated mice deficient in vti1b to study the function of this protein in vivo. vti1b-deficient mice had reduced amounts of syntaxin 8 due to degradation of the syntaxin 8 protein, while the amounts of syntaxin 7 and endobrevin did not change. These data indicate that vti1b is specifically required for the stability of a single SNARE partner. vti1b-deficient mice were viable and fertile. Most vti1b-deficient mice were indistinguishable from wild-type mice and did not display defects in transport to the lysosome. However, 20% of the vti1b-deficient mice were smaller. Lysosomal degradation of an endocytosed protein was slightly delayed in hepatocytes derived from these mice. Multivesicular bodies and autophagic vacuoles accumulated in hepatocytes of some smaller vti1b-deficient mice. This suggests that other SNAREs can compensate for the reduction in syntaxin 8 and for the loss of vti1b in most mice even though vti1b shows only 30% amino acid identity with its closest relative.
In LAMP-2–deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2–deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2–deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation.
Selective disintegration of membrane-enclosed autophagic bodies is a feature of eukaryotic cells not studied in detail. Using a Saccharomyces cerevisiae mutant defective in autophagic-body breakdown, we identified and characterized Aut5p, a glycosylated integral membrane protein. Site-directed mutagenesis demonstrated the relevance of its putative lipase active-site motif for autophagic-body breakdown. aut5Δ cells show reduced protein turnover during starvation and are defective in maturation of proaminopeptidase I. Most recently, by means of the latter phenotype, Aut5p was independently identified as Cvt17p. In this study we additionally checked for effects on vacuolar acidification and detected mature vacuolar proteases, both of which are prerequisites for autophagic-body lysis. Furthermore, biologically active hemagglutinin-tagged Aut5p (Aut5-Ha) localizes to the endoplasmic reticulum (nuclear envelope) and is targeted to the vacuolar lumen independent of autophagy. In pep4Δ cells immunogold electron microscopy located Aut5-Ha at ∼50-nm-diameter intravacuolar vesicles. Characteristic missorting in vps class E and fab1Δ cells, which affects the multivesicular body (MVB) pathway, suggests vacuolar targeting of Aut5-Ha similar to that of the MVB pathway. In agreement with localization of Aut5-Ha at intravacuolar vesicles in pep4Δ cells and the lack of vacuolar Aut5-Ha in wild-type cells, our pulse-chase experiments clearly indicated that Aut5-Ha degradation with 50 to 70 min of half-life is dependent on vacuolar proteinase A.