HDAC6 controls autophagosome maturation essential for ubiquitin‐selective quality‐control autophagy

Autophagy is primarily considered a non‐selective degradation process induced by starvation. Nutrient‐independent basal autophagy, in contrast, imposes intracellular QC by selective disposal of aberrant protein aggregates and damaged organelles, a process critical for suppressing neurodegenerative diseases. The molecular mechanism that distinguishes these two fundamental autophagic responses, however, remains mysterious. Here, we identify the ubiquitin‐binding deacetylase, histone deacetylase‐6 (HDAC6), as a central component of basal autophagy that targets protein aggregates and damaged mitochondria. Surprisingly, HDAC6 is not required for autophagy activation; rather, it controls the fusion of autophagosomes to lysosomes. HDAC6 promotes autophagy by recruiting a cortactin‐dependent, actin‐remodelling machinery, which in turn assembles an F‐actin network that stimulates autophagosome–lysosome fusion and substrate degradation. Indeed, HDAC6 deficiency leads to autophagosome maturation failure, protein aggregate build‐up, and neurodegeneration. Remarkably, HDAC6 and F‐actin assembly are completely dispensable for starvation‐induced autophagy, uncovering the fundamental difference of these autophagic modes. Our study identifies HDAC6 and the actin cytoskeleton as critical components that define QC autophagy and uncovers a novel regulation of autophagy at the level of autophagosome–lysosome fusion.     

Global analysis of cellular protein flux quantifies the selectivity of basal autophagy

In eukaryotic cells, the macroautophagy pathway has been implicated in the degradation of long-lived proteins and damaged organelles. Although it has been demonstrated that macroautophagy can selectively degrade specific targets, its contribution to the basal turnover of cellular proteins had previously not been quantified on proteome-wide scales. In a recent study, we utilized dynamic proteomics to provide a global comparison of protein half-lives between wild-type and autophagy-deficient cells. Our results indicated that in quiescent fibroblasts, macroautophagy contributes to the basal turnover of a substantial fraction of the proteome. However, the contribution of macroautophagy to constitutive protein turnover is variable within the proteome. The methodology outlined in the study provides a global strategy for quantifying the selectivity of basal macroautophagy.     

Uptake and degradation of cytoplasmic RNA by hepatic lysosomes. Quantitative relationship to RNA turnover.

Past evidence has suggested that the lysosomal pathway is an important site of cytoplasmic RNA degradation in the hepatic parenchymal cell (Lardeux, B. R., Heydrick, S. J., and Mortimore, G. E. (1987) J. Biol. Chem. 262, 14507-14519). We now provide additional support for this notion by quantitating degradable RNA in lysosomes and correlating its pool size with hepatic RNA degradation. Rat livers, previously labeled with [6-14C]orotic acid, were perfused with graded levels of amino acids over the full range of induced autophagy; RNA degradation was determined from [14C]cytidine release. Close correspondence between the marker beta-acetylglucosaminidase and the breakdown of RNA to cytidine in subcellular fractions indicated that the lysosome was the main site of catabolism, a conclusion supported by the fact that degradation was enhanced when external pH was lowered from 7 to 6. Although [14C]cytidine was also released in homogenates by the action of natural ribonucleases on cytosolic RNA, this source was eliminated by unlabeled exogenous RNA. The size of the degradable RNA pool in lysosomes, determined from the total release of cytidine in homogenates, correlated directly with rates of hepatic RNA degradation over the full range of basal and induced degradation. A direct correlation was also seen between RNA degradation and cytidine pools within lysosomal particles. Because cytosolic cytidine was not taken up by lysosomes under these conditions, the pool could only have arisen from the breakdown of intralysosomal RNA. As determined by cytidine production, these findings support the view that the lysosomal-vacuolar system is the main, if not sole, site of induced and basal RNA degradation in liver.     

Degradation of protein translation machinery by amino acid starvation-induced macroautophagy

Macroautophagy is regarded as a nonspecific bulk degradation process of cytoplasmic material within the lysosome. However, the process has mainly been studied by nonspecific bulk degradation assays using radiolabeling. In the present study we monitor protein turnover and degradation by global, unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways in stress-induced macroautophagy.     

Regulation of membrane protein degradation by starvation-response pathways

The multivesicular body (MVB) pathway delivers membrane proteins to the lumen of the vacuole/lysosome for degradation. The resulting amino acids are transported to the cytoplasm for reuse in protein synthesis. Our study shows that this amino acid recycling system plays an essential role in the adaptation of cells to starvation conditions. Cells respond to amino acid starvation by upregulating both endocytosis and the MVB pathway, thereby providing amino acids through increased protein turnover. Our data suggest that increased Rsp5-dependent ubiquitination of membrane proteins and a drop in Ist1 levels, a negative regulator of endosomal sorting complex required for transport (ESCRT) activity, cause this response. Furthermore, we found that target of rapamycin complex 1 (TORC1) and a second, unknown nutrient-sensing system are responsible for the starvation-induced protein turnover. Together, the data indicate that protein synthesis and turnover are linked by a common regulatory system that ensures adaptation and survival under nutrient-stress conditions.     

The novel estrogen-induced gene EIG121 regulates autophagy and promotes cell survival under stress

We previously identified a novel estrogen-induced gene, EIG121, as being differentially regulated in endometrioid and nonendometrioid endometrial carcinoma. The function of EIG121 was unknown. Using a tetracycline-inducible system, we found that overexpression of EIG121, but not of LacZ, caused a profound suppression of cell growth. Subcellular fractionation and immunofluroscent labeling indicated that EIG121 was a transmembrane protein localized in the plasma membrane-late endosome–lysosome compartments. Deletion of the putative transmembrane domain abolished the membrane association. In cells overexpressing EIG121, cytoplasmic vacuoles accumulated after EIG121 induction, and the autophagosome marker LC3 translocated into punctuate, dot-like structures. Electron microscopy revealed that in cells overexpressing EIG121, autophagosomes were markedly increased. Overexpression of EIG121 also increased the cells containing acidic vesicles and induced lysosomal degradation of long-lived proteins. In MCF-7 cells, both EIG121 and LC3 were rapidly degraded by a lysosomal mechanism after starvation. Knockdown of EIG121 blocked starvation-induced LC3 degradation. By itself, knockdown of EIG121 did not affect cell survival. When combined with starvation or cytotoxic agents, EIG121 knockdown greatly increased apoptosis. Our results suggest that EIG121 is associated with the endosome–lysosome compartments and may have an important role in autophagy. Under unfavorable conditions such as starvation and exposure to cytotoxic agents, EIG121 may protect cells from cell death by upregulating the autophagy pathway.     

Degradation of intracellular protein in muscle. Lysosomal response to modified proteins and chloroquine

We previously showed that radioactive N-ethylmaleimide injected intramuscularly reacts with actomyosin and other muscle proteins and that a transfer of these modified proteins to lysosome-rich large granules was associated with their degradation (Gerard, K. W., and Schneider, D. L., (1979) J. Biol. Chem. 254, 11798-11805). We now show that muscle cells, when challenged by an increase in proteins modified with N-ethylmaleimide, can increase degradation by increasing the activities of enzymes involved in protein turnover. Cathepsin B activity increased 2-fold 36 h after injection of N-ethylmaleimide. In contrast, non-lysosomal proteolytic enzymes, calcium-dependent protease, and leucine aminopeptidase, did not significantly increase. Lysosomes are also involved in the degradation of normal muscle proteins labeled with [3H]leucine. Treatment with chloroquine, a known inhibitor of lysosome function, resulted in an inhibition of protein degradation, in an increase of the muscle protein content, and in the accumulation of radioactive proteins in lysosomal fractions. Chloroquine treatment for 2 days led to a 270% increase in cathepsin B and a 160% increase in lysosomal ATPase activities, but only a 30% increase in neutral proteinase activities. These results indicate a role for lysosomes in regulation of protein turnover in muscle.     

The ATP dependence of the degradation of short- and long-lived proteins in growing fibroblasts

To characterize the system(s) responsible for degradation of short-lived and long-lived proteins in mammalian cells, we compared the concentrations of ATP required for the degradation of these classes of proteins in growing hamster fibroblasts. By treating CHEF-18 cells with increasing concentrations of dinitrophenol and 2-deoxyglucose, it was possible to reduce their steady-state ATP content by different amounts (up to 98%). These treatments caused a rapid decrease in the degradation of both short- and long-lived proteins. Removal of the inhibitors led to a prompt restoration of ATP and proteolysis. As ATP content fell below normal levels (about 3.1 mM), rates of proteolysis decreased in a graded biphasic fashion. Reduction in ATP by up to 90% (as may occur in anoxia or injury) decreased proteolysis up to 50%; and with further loss of ATP, protein breakdown fell more sharply. Degradation of both classes of proteins was inhibited by 80% when ATP levels were reduced by 98%. The levels of ATP required for the breakdown of short- and long-lived proteins were indistinguishable. Protein synthesis was much more sensitive to a decrease in ATP content than protein breakdown and fell by 50% when ATP was reduced by only 15%. Chloroquine, an inhibitor of lysosome function, did not reduce the degradation of either class of proteins in growing cells, but it did inhibit the enhanced degradation of long-lived proteins upon removal of serum (in accord with previous studies). Thus, in growing fibroblasts, an ATP-dependent nonlysosomal process appears responsible for the hydrolysis of both short- and long-lived proteins.     

Abl kinases regulate autophagy by promoting the trafficking and function of lysosomal components

Autophagy is a lysosome-dependent degradative pathway that regulates the turnover of intracellular organelles, parasites, and long-lived proteins. Deregulation of autophagy results in a variety of pathological conditions, but little is known regarding the mechanisms that link normal cellular and pathological signals to the regulation of distinct stages in the autophagy pathway. Here we uncover a novel role for the Abl family kinases in the regulation of the late stages of autophagy. Inhibition, depletion, or knockout of the Abl family kinases, Abl and Arg, resulted in a dramatic reduction in the intracellular activities of the lysosomal glycosidases alpha-galactosidase, alpha-mannosidase and neuraminidase. Inhibition of Abl kinases also reduced the processing of the precursor forms of cathepsin D and cathepsin L to their mature, lysosomal forms, which coincided with the impaired turnover of long-lived cytosolic proteins and accumulation of autophagosomes. Furthermore, defective lysosomal degradation of long-lived proteins in the absence of Abl kinase signaling was accompanied by a perinuclear redistribution of lysosomes and increased glycosylation and stability of lysosome-associated membrane proteins, which are known to be substrates for lysosomal enzymes and play a role in regulating lysosome mobility. Our findings reveal a role for Abl kinases in the regulation of late-stage autophagy and have important implications for therapies that employ pharmacological inhibitors of the Abl kinases.     

Coronavirus NSP6 restricts autophagosome expansion

Autophagy is a cellular response to starvation that generates autophagosomes to carry long-lived proteins and cellular organelles to lysosomes for degradation. Activation of autophagy by viruses can provide an innate defense against infection, and for (+) strand RNA viruses autophagosomes can facilitate assembly of replicase proteins. We demonstrated that nonstructural protein (NSP) 6 of the avian coronavirus, infectious bronchitis virus (IBV), generates autophagosomes from the ER. A statistical analysis of MAP1LC3B puncta showed that NSP6 induced greater numbers of autophagosomes per cell compared with starvation, but the autophagosomes induced by NSP6 had smaller diameters compared with starvation controls. Small diameter autophagosomes were also induced by infection of cells with IBV, and by NSP6 proteins of MHV and SARS and NSP5, NSP6, and NSP7 of arterivirus PRRSV. Analysis of WIPI2 puncta induced by NSP6 suggests that NSP6 limits autophagosome diameter at the point of omegasome formation. IBV NSP6 also limited autophagosome and omegasome expansion in response to starvation and Torin1 and could therefore limit the size of autophagosomes induced following inhibition of MTOR signaling, as well as those induced independently by the NSP6 protein itself. MAP1LC3B-puncta induced by NSP6 contained SQSTM1, which suggests they can incorporate autophagy cargos. However, NSP6 inhibited the autophagosome/lysosome expansion normally seen following starvation. Taken together the results show that coronavirus NSP6 proteins limit autophagosome expansion, whether they are induced directly by the NSP6 protein, or indirectly by starvation or chemical inhibition of MTOR signaling. This may favor coronavirus infection by compromising the ability of autophagosomes to deliver viral components to lysosomes for degradation.     

Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice

Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.     

Coronavirus NSP6 restricts autophagosome expansion

Autophagy is a cellular response to starvation that generates autophagosomes to carry long-lived proteins and cellular organelles to lysosomes for degradation. Activation of autophagy by viruses can provide an innate defense against infection, and for (+) strand RNA viruses autophagosomes can facilitate assembly of replicase proteins. We demonstrated that nonstructural protein (NSP) 6 of the avian coronavirus, infectious bronchitis virus (IBV), generates autophagosomes from the ER. A statistical analysis of MAP1LC3B puncta showed that NSP6 induced greater numbers of autophagosomes per cell compared with starvation, but the autophagosomes induced by NSP6 had smaller diameters compared with starvation controls. Small diameter autophagosomes were also induced by infection of cells with IBV, and by NSP6 proteins of MHV and SARS and NSP5, NSP6, and NSP7 of arterivirus PRRSV. Analysis of WIPI2 puncta induced by NSP6 suggests that NSP6 limits autophagosome diameter at the point of omegasome formation. IBV NSP6 also limited autophagosome and omegasome expansion in response to starvation and Torin1 and could therefore limit the size of autophagosomes induced following inhibition of MTOR signaling, as well as those induced independently by the NSP6 protein itself. MAP1LC3B-puncta induced by NSP6 contained SQSTM1, which suggests they can incorporate autophagy cargos. However, NSP6 inhibited the autophagosome/lysosome expansion normally seen following starvation. Taken together the results show that coronavirus NSP6 proteins limit autophagosome expansion, whether they are induced directly by the NSP6 protein, or indirectly by starvation or chemical inhibition of MTOR signaling. This may favor coronavirus infection by compromising the ability of autophagosomes to deliver viral components to lysosomes for degradation.     

Rates of protein turnover in vivo and in vitro in ventricular muscle of hearts from fed and starved rats.

Starvation of 300 g rats for 3 days decreased ventricular-muscle total protein content and total RNA content by 15 and 22% respectively. Loss of body weight was about 15%. In glucose-perfused working rat hearts in vitro, 3 days of starvation inhibited rates of protein synthesis in ventricles by about 40-50% compared with fed controls. Although the RNA/protein ratio was decreased by about 10%, the major effect of starvation was to decrease the efficiency of protein synthesis (rate of protein synthesis relative to RNA). Insulin stimulated protein synthesis in ventricles of perfused hearts from fed rats by increasing the efficiency of protein synthesis. In vivo, protein-synthesis rates and efficiencies in ventricles from 3-day-starved rats were decreased by about 40% compared with fed controls. Protein-synthesis rates and efficiencies in ventricles from fed rats in vivo were similar to values in vitro when insulin was present in perfusates. In vivo, starvation increased the rate of protein degradation, but decreased it in the glucose-perfused heart in vitro. This contradiction can be rationalized when the effects of insulin are considered. Rates of protein degradation are similar in hearts of fed animals in vivo and in glucose/insulin-perfused hearts. Degradation rates are similar in hearts of starved animals in vivo and in hearts perfused with glucose alone. We conclude that the rates of protein turnover in the anterogradely perfused rat heart in vitro closely approximate to the rates in vivo in absolute terms, and that the effects of starvation in vivo are mirrored in vitro.     

Quantifying the contribution of defective ribosomal products to antigen production: a model-based computational analysis

Antigenic peptides (epitopes) presented on the cell surface by MHC class I molecules derive from proteolytic degradation of endogenous proteins. Some recent studies have proposed that the majority of epitopes stem from so-called defective ribosomal products (DRiPs), i.e., freshly synthesized proteins that are unable to adopt the native conformation and thus undergo immediate degradation. However, a reliable computational analysis of the data underlying this hypothesis was lacking so far. Therefore, we have applied kinetic modeling to derive from existing kinetic data (Princiotta et al. 2003, Immunity 18, 343-354) the rates of the major processes involved in the cellular protein turnover and MHC class I-mediated Ag presentation. From our modeling approach, we conclude that in these experiments 1) the relative share of DRiPs in the total protein synthesis amounted to approximately 10% thus being much lower than reported so far, 2) DRiPs may become the decisive source of epitopes within an early phase after onset of the synthesis of a long-lived (e.g., virus derived) protein, and 3) inhibition of protein synthesis by the translation inhibitor cycloheximide appears to be paralleled with an instantaneous decrease of protein degradation down to approximately 1/3 of the normal value.     

Macromolecular Synthesis and Degradation in Arthrobacter During Periods of Nutrient Deprivation

Cells of Arthrobacter atrocyaneus and A. crystallopoietes, harvested during their exponential phase, were starved in 0.03 M phosphate buffer (pH 7.0) for 28 days. During this time, the cells maintained 90 to 100% viability. Experimental results were similar for both organisms. Total cellular deoxyribonucleic acid was maintained. Measurable degradation rates for deoxyribonucleic acid as determined by radioisotope techniques were not observed, and only during the initial hours of starvation could a synthetic rate be determined. Total ribonucleic acid levels remained stable for the first 24 h of starvation, after which slow, continuous loss of orcinol-reactive material occurred. Synthetic and degradative rates of ribonucleic acid, as determined by radioisotope techniques, dropped quickly at the onset of starvation. Constant basal rates were attained after 24 h. In A. atrocyaneus, total cell protein was degraded continuously from the onset of starvation. In A. crystallopoietes, total cell protein remained stable for the first 24 h, after which slow continuous loss occurred. After 28 days, the total protein per cell was similar for both organisms. In the first week, amino acid pools stabilized at about 50% of the values characteristic of growth. Rates of degradation of protein decreased rapidly for the first 24 h for both organisms, but leveled to a constant basal rate thereafter. Rates of new protein synthesis dropped during the first 24 h and by 48 h achieved a constant basal rate.     

Intracellular protein degradation in Neurospora crassa.

In exponentially growing cultures of Neurospora crassa, the basal rate of protein degradation increases as the constant of the rate of growth decreases, so that in slow growing cells (mu = 0.13) the rate of protein degradation is about 25% of the rate of protein accumulation. During glucose starvation and shift-down transition of growth, the rate of protein degradation is greatly enhanced, and a moderate reduction (about 30%) of the ATP level is observed. Treatment of glucose-starved cells with 2-deoxyglucose reduces the ATP content by 70% and blocks protein degradation. The addition of cycloheximide, given at the onset of glucose starvation, prevents the enhancement of protein degradation; instead cycloheximide is without effect if added when proteolysis has already started. At a supraoptimal temperature (42 degrees C) the basal rate of protein degradation is not stimulated, contrary to the behavior observed in bacteria. Guanosine nucleotides, which appear to have a regulatory role for protein degradation in bacteria, are not found in N. crassa.     

In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker

Macroautophagy mediates the bulk degradation of cytoplasmic components. It accounts for the degradation of most long-lived proteins: cytoplasmic constituents, including organelles, are sequestered into autophagosomes, which subsequently fuse with lysosomes, where degradation occurs. Although the possible involvement of autophagy in homeostasis, development, cell death, and pathogenesis has been repeatedly pointed out, systematic in vivo analysis has not been performed in mammals, mainly because of a limitation of monitoring methods. To understand where and when autophagy occurs in vivo, we have generated transgenic mice systemically expressing GFP fused to LC3, which is a mammalian homologue of yeast Atg8 (Aut7/Apg8) and serves as a marker protein for autophagosomes. Fluorescence microscopic analyses revealed that autophagy is differently induced by nutrient starvation in most tissues. In some tissues, autophagy even occurs actively without starvation treatments. Our results suggest that the regulation of autophagy is organ dependent and the role of autophagy is not restricted to the starvation response. This transgenic mouse model is a useful tool to study mammalian autophagy.     

Regulation of connexin degradation as a mechanism to increase gap junction assembly and function

Connexins, the integral membrane protein constituents of gap junctions, are degraded at a rate (t(12) = 1.5-5 h) much faster than most other cell surface proteins. Although the turnover of connexins has been shown to be sensitive to inhibitors of either the lysosome or of the proteasome, how connexins are targeted for degradation and whether this process can be regulated to affect intercellular communication is unknown. We show here that reducing connexin degradation with inhibitors of the proteasome (but not with lysosomal blockers) is associated with a striking increase in gap junction assembly and intercellular dye transfer in cells inefficient in both processes under basal conditions. The effect of proteasome inhibitors on wild-type connexin stability, assembly, and function was mimicked by treatment of assembly-inefficient cells with inhibitors of protein synthesis such as cycloheximide. Sensitivity of connexin degradation to cycloheximide, but not to proteasome inhibitors, was abolished when connexins were rendered structurally abnormal by perturbation of essential disulfide bonds or by mutation. Our findings provide the first evidence that intercellular communication can be up-regulated at the level of connexin turnover and that a short-lived protein may be required for conformationally mature connexins to become substrates of proteasomal degradation.     

Regulation of protein synthesis and degradation in L8 myotubes. Effects of serum, insulin and insulin-like growth factors.

We have examined the regulation of protein turnover in rat skeletal myotubes from the L8 cell line. We measured protein synthesis by the rates of incorporation of radiolabelled tyrosine into protein in the presence of a flooding dose of non-radioactive tyrosine. We monitored degradation of proteins labelled with radioactive tyrosine by the release of acid-soluble radioactivity into medium containing excess nonradioactive tyrosine. Extracellular tyrosine pools and intracellular tyrosyl-tRNA equilibrate rapidly during measurements of protein synthesis, and very little reutilization of the radiolabelled tyrosine occurs during degradation measurements. Measured rates of protein synthesis and degradation are constant for several hours, and changes in myotube protein content can be accurately predicted by the measured rates of protein synthesis and degradation. Most of the myotube proteins labelled with radioactive tyrosine for 2 days are degraded, with half-lives (t1/2) of approx. 50 h. A small proportion (less than 2.5%) of the radiolabelled proteins are degraded more rapidly (t1/2 less than 10 h), and, at most, a small proportion (less than 15%) are degraded more slowly (t1/2 greater than 50 h). A variety of agents commonly added to primary muscle cell cultures or to myoblast cell lines (18% Medium 199, 1% chick-embryo extract, antibiotics and antifungal agents) had no effect on rates of protein synthesis or degradation. Horse serum, fetal bovine serum and insulin stimulate protein synthesis and inhibit the degradation of long-lived proteins without affecting the degradation of short-lived proteins. Insulin-like growth factors (IGF)-1 and -2 also stimulate protein synthesis and inhibit protein degradation. The stimulation of protein synthesis and the inhibition of protein degradation are of similar magnitude (a maximum of approx. 2-fold) and display similar sensitivities to a particular anabolic agent. Insulin stimulates protein synthesis and inhibits protein degradation only at supraphysiological doses, whereas IGF-1 and -2 are effective at physiological concentrations. These and other findings suggest that IGFs may be important regulators of skeletal muscle growth during the fetal and early neonatal periods.     

Substrate recognition in selective autophagy and the ubiquitin–proteasome system

Dynamic protein turnover through regulated protein synthesis and degradation ensures cellular growth, proliferation, differentiation and adaptation. Eukaryotic cells utilize two mechanistically distinct but largely complementary systems — the 26S proteasome and the lysosome (or vacuole in yeast and plants) — to effectively target a wide range of proteins for degradation. The concerted action of the ubiquitination machinery and the 26S proteasome ensures the targeted and tightly regulated degradation of a subset of commonly short-lived cellular proteins. Autophagy is a distinct degradation pathway, which transports a highly heterogeneous set of cargos in dedicated vesicles, called autophagosomes, to the lysosome. There the cargo becomes degraded and its molecular building blocks are recycled. While general autophagy randomly engulfs portions of the cytosol, selective autophagy employs dedicated cargo adaptors to specifically enrich the forming autophagosomes for a certain type of cargo as a response to various intra- or extracellular signals. Selective autophagy targets a wide range of cargos including long-lived proteins and protein complexes, organelles, protein aggregates and even intracellular microbes. In this review we summarize available data on cargo recognition mechanisms operating in selective autophagy and the ubiquitin–proteasome system (UPS), and emphasize their differences and common themes. Moreover, we derive general regulatory principles underlying cargo recognition in selective autophagy, and describe the system-wide crosstalk between these two cellular protein degradation systems. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.