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Reciprocal regulation of chaperone-mediated autophagy and the circadian clock

Nature Cell Biology volume 23, pages 1255–1270 (2021)Cite this article

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Abstract

Circadian rhythms align physiological functions with the light–dark cycle through oscillatory changes in the abundance of proteins in the clock transcriptional programme. Timely removal of these proteins by different proteolytic systems is essential to circadian strength and adaptability. Here we show a functional interplay between the circadian clock and chaperone-mediated autophagy (CMA), whereby CMA contributes to the rhythmic removal of clock machinery proteins (selective chronophagy) and to the circadian remodelling of a subset of the cellular proteome. Disruption of this autophagic pathway in vivo leads to temporal shifts and amplitude changes of the clock-dependent transcriptional waves and fragmented circadian patterns, resembling those in sleep disorders and ageing. Conversely, loss of the circadian clock abolishes the rhythmicity of CMA, leading to pronounced changes in the CMA-dependent cellular proteome. Disruption of this circadian clock/CMA axis may be responsible for both pathways malfunctioning in ageing and for the subsequently pronounced proteostasis defect.

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Fig. 1: Components of the molecular clock are degraded in lysosomes via CMA.
Fig. 2: Blockage of CMA disrupts the molecular clock.
Fig. 3: CMA-defective mice display disturbances in circadian patterns.
Fig. 4: CMA displays BMAL1-dependent circadian activity in liver.
Fig. 5: BMAL1 regulates circadian CMA activity in liver at the transcriptional level.
Fig. 6: Circadian cycling of CMA activity is tissue specific.
Fig. 7: Cyclic changes in the lysosomal proteome and impact of CMA blockage on lysosomal-resident proteins.
Fig. 8: CMA contributes to circadian remodelling of the proteome.

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Data availability

The proteomic data are deposited at ProteomeXchange via the PRIDE partner repository with the dataset identifier PXD019704. There are no restrictions on availability of data presented in this study. Source data are provided with this paper.

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Acknowledgements

We thank S. Tiano for excellent technical support with animal maintenance and biochemical procedures, J. Madrigal-Matute for early assistance in the analysis of the lysoproteome, and the Analytical Image and Animal Physiology Cores of the Albert Einstein College of Medicine for assistance with image acquisition and analysis and metabolic behaviour measurements. Circadian running wheel procedures were performed with assistance from The Neurobehavior Testing Core at UPenn/ITMAT and Intellectual and IDDRC at CHOP/Penn. This work was supported by grants from the National Institutes of Health (NIH) AG021904, AG054108, DK098408 (to A.M.C.), RF1AG043517 (to R.S.), AG031782 (to A.M.C., R.S. and F.M.) and the generous support of the Rainwaters Foundation and the JPB Foundation (to A.M.C.), the Glenn Foundation (to A.M.C. and F.M.), the Ikerbasque, Basque Foundation for Science, Bilbao, Spain (to J.M.) and U54 HD086984 (U Penn/IDDRC core) and AG038072 (Einstein Animal Physiology Core). Y.R.J. was supported by NIH training grant T32GM007491, G.J.K. by NIH training grants T32GM007288 and T32GM007491, and M.J. by NIH training grant T32HL14445. The CNIO Proteomics Unit lab is a member of Proteored, PRB3 and is supported by grant PT17/0019, of the PE I+D+i 2013–2016, funded by ISCIII and ERDF.

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Author notes

  1. These authors contributed equally: Yves R. Juste, Susmita Kaushik.

Authors and Affiliations

  1. Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY, USA

    Yves R. Juste, Susmita Kaushik, Mathieu Bourdenx, Antonio Diaz, Kristen Lindenau, Gregory J. Krause, Maryam Jafari, Rajat Singh & Ana Maria Cuervo

  2. Institute for Aging Studies, Albert Einstein College of Medicine, Bronx, NY, USA

    Yves R. Juste, Susmita Kaushik, Mathieu Bourdenx, Antonio Diaz, Kristen Lindenau, Gregory J. Krause, Maryam Jafari, Rajat Singh, Fernando Macian & Ana Maria Cuervo

  3. Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA

    Ranee Aflakpui, Sanmay Bandyopadhyay, Vincent Tu & Fernando Macian

  4. Proteomic Unit, Spanish National Cancer Research Center (CNIO) Proteored-ISCIII, Madrid, Spain

    Fernando Garcia & Javier Muñoz

  5. Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

    Rajat Singh & Ana Maria Cuervo

  6. Biocruces Bizkaia Health Research Institute, Barakaldo, Spain

    Javier Muñoz

  7. Ikerbasque, Basque Foundation for Science, Bilbao, Spain

    Javier Muñoz

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  1. Yves R. Juste
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Contributions

Y.R.J. conceived, designed and performed most of the biochemical, molecular biology and image-based experiments, analysed the data, prepared the first draft of the manuscript, and revised the final version; S.K. performed biochemical experiments, analysed the data, prepared the revised final version and proofread and edited the manuscript; M.B. performed immunofluorescence of the SCN and computational analysis; R.A. performed analysis of proteasome activities; S.B. performed the Lamp2a promoter expression analysis, F.G. carried out the proteomic procedures and contributed to data interpretation; A.D. generated animal colonies and assisted with their characterization; K.L. assisted with cell culture and cell transfection; V.T. performed the point mutagenesis; G.J.K. and M.J. performed experiments with ageing mice; R.S. provided samples of the iTAD experiments; J.M. led, designed and interpreted the proteomic analysis and edited the manuscript; F.M. directed the analysis of the transcriptional regulation of BMAL1 on CMA and assisted with data interpretation; A.M.C. conceived and directed the study, contributed to manuscript writing and edited the final version of the manuscript.

Corresponding author

Correspondence to Ana Maria Cuervo.

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Competing interests

A.M.C. is the founder and serves in the Scientific Board of Selphagy (a programme under LifeBiosciences), and she consults for Generian Pharmaceuticals and Cognition Therapeutics. The other authors declare no competing interests.

Additional information

Peer review information Nature Cell Biology thanks Salvador Benitah, Masaaki Komatsu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Circadian proteins display properties of bona fide CMA substrates.

a. Representative immunoblot for LAMP2A from rat liver lysosomes immunoprecipitated for BMAL1 (left), CLOCK (middle), or REVERBα (right). The heavy chain (HC) of IgG used in the immunoprecipitation is shown. Input is 5% amount used for IP. b. Representative immunoblots for HSC70 from wild-type (WT) or LAMP2A knockout (L2AKO) cell lysates immunoprecipitated for BMAL1 or CLOCK. Heavy chain (HC) for IgG used in the immunoprecipitation is shown. Input is 5% concentration used for IP. Right, quantification of HSC70 normalized by the amount of BMAL1 or CLOCK immunoprecipitated. An increase in the amount of CMA substrates bound at a given time to HSC70, similar to the one observed here for BMAL1 and CLOCK, has previously been described upon blockage of CMA. n=3 independent experiments. Individual values (b) and mean+s.e.m are shown. Unpaired two-tailed t-test was used, and differences were significant for *p<0.05. c. Representative immunoblot of competition of BMAL1 lysosomal uptake by ribonuclease A (RNase A). Left: representative immunoblot. Right: effect of increasing concentrations of RNase A on binding and uptake of 20ng of BMAL1. Numerical source data, statistics and exact p values and unprocessed blots are available as source data.

Source data

Extended Data Fig. 2 Circadian lysosomal and proteasomal protein degradation in liver.

a. Chymotrypsin-like (CTL) and caspase-like (CSP) activities of the 20S and 26S proteasome in livers from mice injected with saline (Sal) or leupeptin (Leup; 40mg/kg b.w.) 3h before tissue collection. Area under the curve after discounting residual MG115-resistant activity (left) and time-course kinetics (right) are shown. n=4 (CTL), 7 (CSP) mice. b. Top: Immunoblot from livers from mice treated as in a collected at the indicated Zeitgeber times (ZT). Ponceau staining is shown as loading control. Bottom: Levels of K48-polyubiquitinated proteins (left) and LC3-II (right) relative to ZT2-5 saline injected. n=3 mice per ZT and treatment. White and black strips throughout the figure denote light or dark period, respectively. c. Proteolytic activity of liver lysosomes from mice injected with MG262 in 60% dimethyl sulfoxide (DMSO) or only DMSO (control; CTR) and incubated with a pool of radiolabeled proteins with or without leupeptin. n=3 mice per treatment. d. Left: Representative immunoblot of homogenate of mice injected (+) or not (-) with leupeptin (leup) as in a. Right: LC3-II flux (fold increase upon leupeptin injection). n=3 mice per ZT. e-g. Immunoblot of liver homogenates (Hom), lysosomes (Lys) active (+) or not (-) for CMA, ER, and cytosol (Cyt) (e), CMA+ lysosomes (f) and CMA+ and CMA (-) lysosomes (g) isolated from mice treated as in a. Loading controls for f are in e. h. Immunoblot of rat liver fractions at ZT17 to compare with ZT5 (Fig. 1b). i. Immunoblot of liver homogenates from mice treated as in c. UB-K48 is used as a control for efficacy of MG262 injection. j. Proteasome degradation of PER1 and CRY2 (>1.5 fold increase upon MG262 injection) at each time. n=4 mice per ZT. Dotted line: no degradation. Individual values (a-d,j) and mean+s.e.m are shown. Unpaired t-test (a left) and Two-way ANOVA followed by Sidak (a right, b and c) or Bonferroni’s (c, d, j) multiple comparisons post-hoc tests were used Differences were significant for *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. ns = not significant. Numerical source data, statistics and exact p values and unprocessed blots are available as source data.

Source data

Extended Data Fig. 3 Lysosomal degradation of clock proteins is dependent on CMA but independent of the nutritional status.

a. Immunoblot of liver homogenates from LAMP2A knockout mice (L2AKO) intraperitoneally injected (i.p.) with saline (Sal) or a single dose of leupeptin 3h before tissue collection at the indicated circadian times (ZT). b. Lysosomal flux for the indicated circadian proteins calculated as the increase in >1.5 folds above the dotted line (value 1) upon leupeptin injection. n=3 mice per ZT. c. Immunoblot of livers from WT and L2AKO mice treated as in a and collected at ZT(20-23), time of maximal BMAL1 and CLOCK lysosomal degradation, and run in the same membrane for comparative purposes. d. Lysosomal flux for the indicated proteins in WT and L2AKO mice livers from blots in Extended Data Fig. 2f and 3a,c. n=3 mice per ZT and genotype. A lane with the same sample in all gels was used for normalization across membranes. Note that PER1 was not detected in WT lysosomes (Extended Data Fig. 2g). e. Immunoblot for the indicated proteins in CMA-active lysosomes (Lys CMA+) and CMA-inactive lysosomes (Lys CMA-) from mice fed ad libitum or starved for 24h (Stv) and injected or not with leupeptin. A representative immunoblot is shown (the experiment was replicated 3 times). f,g. Immunoblot for HSC70 (f) and LAMP2A (g) of BMAL1 immunoprecipitated (IP) from liver homogenates of mice fed ad libitum (AL) or maintained in an isocaloric twice-a-day feeding (iTAD) during the light time for 6 months collected at ZT8 and ZT20. The experiment was repeated 3 times. White and black strips denote light or dark period, respectively. Individual values and mean+s.e.m are shown. Two-way ANOVA followed by Bonferroni’s multiple comparisons post-hoc test was used to determine differences in protein flux at each time in L2AKO mice (b) and differences between genotypes (d). Differences in flux between both ZT are shown in the legend and between genotypes on top of the bars. Absolute protein levels are included in the raw data file. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. ns = not significant. Numerical source data, statistics and exact p values and unprocessed blots are available as source data.

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Extended Data Fig. 4 Properties of CMA targeted BMAL1.

a. Immunoblot for BMAL1 phosphorylated on serine 42 (pBMAL1S42) in wild-type (WT, W) and LAMP2A knockout mice (L2AKO, L) livers at the indicated Zeitgeber times (ZT). Total BMAL1 and actin immunoblots from Fig. 2a are shown here for comparison. Arrows: BMAL1 bands of different electrophoretic mobility. All membranes contained a common sample for normalization across membranes. White and black strips throughout the figure denote light or dark period, respectively. b. Levels of the two bands of total (top) and pBMAL1S42 (bottom) in WT (left) and L2AKO mice (right) from immunoblots as in a. Values are percentage of total BMAL1 contributed by each band. n=3 mice per ZT. c. Immunoblot for total BMAL1, pBMAL1S42 and BMAL1 acetylated on lysine 538 (AcBMAL1K538) in mouse liver homogenates (Hom) and lysosomes (Lys) active (+) or not (-) for CMA at the time of maximal BMAL1 lysosomal degradation. LAMP2A and HSC70 from the same fractions are shown on the right. d. Immunoblot of nuclear (left) and cytosolic fractions (right) from WT and L2AKO mice livers collected at the indicated Zeitgeber times (ZT), n=3 mice per ZT and genotype. Actin is shown as cytosolic loading control and Histone 3 as marker of the nuclear fraction. e, f. Levels of the top (left) and bottom (right) bands of BMAL1 (e) and of CLOCK (f) shown in d. Values are folds of ZT11 WT (arbitrary value of 1). n=3 mice per ZT and genotype in e and f. g. Immunoblot for total BMAL1 and pBMAL1S42 in fractions from synchronized NIH3T3 cells cultured or not with leptomycin to block nuclear export and (+) ammonium chloride and leupeptin (N/L) to block lysosomal degradation. Right: higher exposure of lysosome lanes. h. Lysosomal degradation (fold increase upon N/L) of the top and bottom BMAL1 (top) and pBMAL1S42 bands (bottom) in cells in g. Red dotted line: no degradation. n=3 mice per treatment. Individual values (e,f,h) and mean+s.e.m are shown. Two-way ANOVA followed by Bonferroni’s multiple comparisons post-hoc test was used. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Numerical source data, statistics and exact p values and unprocessed blots are available as source data.

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Extended Data Fig. 5 Expression of BMAL1 protein mutated in the CMA-targeting motifs.

a. Immunoblot for His6 tag of NIH3T3 cells stably expressing His6-tagged BMAL1 wild-type (WT) or mutated in both CMA-targeting motifs (DM) at the indicated times after addition of cycloheximide (CHX) to stop protein synthesis. Ponceau is shown as loading control. b. Quantification of BMAL1 in experiments as in a. Values are expressed as fraction of the initial BMAL1 remaining at each time. n=3 independent experiments. c. Immunofluorescence for HSC70 (green) and His6 tag (red) in NIH3T3 cells stably expressing WT and DM BMAL1. Merged channels and colocalization mask are shown. Insets: boxed areas at higher magnification. d. Fraction of His6-BMAL1 colocalizing with HSC70 (Mander’s Coefficient - M1), n=3 independent experiments. e. Co-immunoprecipitation (IP) of HSC70 with BMAL1 in the same cells. Input and flow through (FT) are also shown. f. Amount of HSC70 co-immunoprecipitated with BMAL1 expressed as folds that in WT (arbitrary value of 1), n=3 independent experiments. g. Immunofluorescence for His6 tag in cells as in c. Image shows pseudocolor by gradient intensity. h. Intensity of nuclear BMAL1 in images as in g. n=3 independent experiments (55 cells were quantified per experiment and the mean value of intensity in each experiment was used for statistics). i-l. Immunoblot for CLOCK and CRY1 in lysosomes active (+) or not (-) for CMA isolated from livers of WT or BMAL1 knockout mice (iBKO) at the indicated CT times (i). Quantification of the levels of both proteins in CMA+ (j) and CMA- (k) lysosomes and in the four fractions (l) together for comparative purposes from fractions as the ones in I. Values are expressed as fold those in fractions from CT5 WT. n=3 mice per CT and genotype (in j, k and l). Individual values (d,f,h) and mean+s.e.m are shown. Two-way ANOVA followed by Bonferroni’s (b, j, k) or Tukey’s (l) post-hoc test for multiple variable comparisons and two-tailed unpaired t-test (d, f, h) were used. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. ns = not significant. Numerical source data, statistics and exact p values and unprocessed blots are available as source data.

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Extended Data Fig. 6 Free running behaviour and metabolic characteristics of CMA-deficient mice.

a. Average activity onset relative to light offset (left) and variability of activity onset (right) of wild-type (WT) and LAMP-2A knock out mice (L2AKO) under 12:12 light:dark (LD) cycle. n=32 recordings in 4 mice per genotype (left) and n=4 mice per genotype (right). b. Representative actograms of WT and L2AKO mice during continuous light exposure (LL) n=9 mice per genotype. Black and orange or white strips throughout the figure indicate the 12:12 light:dark cycle in which the mice were originally entrained prior to the shift to constant darkness (DD) or constant light (LL). c, d. Average wheel running revolutions per day (c) and strength of rhythmicity (d) of WT and L2AKO mice in continuous dark (DD) or light (LL), n=10 (WT) and 9 (L2AKO) mice in c and d. e. Representative chi-square periodogram of WT and L2AKO mice in LL. The mean period is in red. n=9 mice per genotype. Significance level (above green line) with chi-squared threshold of 0.001. Orange dotted line: peak period of L2AKO mice (weaker periodicity than WT). f. Double-plotted actograms of WT and L2AKO mice with moderate (1) or severe (2) disruption of circadian properties at the indicated light conditions. Discontinuous lines: corrective shift in response to continuous darkness in WT (black) or L2AKO mice (red). Bottom: BMAL1 immunostaining of SCN at ZT17 in the mice of the actograms shown on top. g. Average intensity per area of BMAL1 staining in SCN of WT and L2AKO mice separated by the severity of their actogram changes, n=3 mice per group. h. Average value during the LD cycles or throughout a 24h period (total) of O2 consumption, CO2 production and energy expenditure (EE) in WT or L2AKO mice. n=4 mice per genotype. i. Median body heat production, VO2 consumption and CO2 production plots from WT and L2AKO mice, n=4 mice per genotype, subjected to alternating light/dark cycles each 2 days (left) or continuous light for 8 days (right). Data is single plotted in two-day intervals. Individual values and mean+s.e.m are shown. Unpaired two-tailed t-test (a, c and d), one-way (g) and two-way (h) ANOVA followed by Bonferroni’s multiple comparisons post-hoc test were used. *p<0.05, **p<0.01, ****p<0.0001. ns = not significant. Numerical source data, statistics and exact p values are available as source data.

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Extended Data Fig. 7 Changes in clock components and in the circadian transcriptome with age in mice with active or inactive CMA activity.

a. Immunoblot for the indicated proteins in livers from 4m and 24m old wild-type (WT) and LAMP2A knockout (L2AKO) mice. n=3 (WT) and 4 (L2AKO) mice per age. Ponceau staining is shown as loading control. b. Levels of the indicated proteins in the same mice obtained by densitometric quantification of immunoblots as in a. Values are expressed as fold levels in 4m old WT (dotted line) n=3 (WT) and 4 (L2AKO) mice per age. Animals were analyzed at ZT2 to catch the end of the nocturnal degradation of BMAL1 and CLOCK and the beginning of the diurnal degradation of REVERBα. c, d. Co-immunoprecipitation of LAMP2A (c) and HSC70 (d) with BMAL1 in livers of 4m and 24m old mice. * sample lost during processing. Experiments were repeated 3 times. e-g. Temporal changes in the mRNA of representative genes involved in carbohydrate (e), lipid metabolism (f) and in cytokine and interleukin signaling (g) shown to undergo reprograming of their circadian expression in old mice in the liver of young WT (W) or L2AKO (L) mice at the indicated ZT times. n=3 mice per ZT and genotype. White and black strips below the graphs denote light and dark period, respectively. Individual values (b) and mean+s.e.m are shown. Two-way ANOVA test followed by Tukey’s multiple comparisons post-hoc test was used in b, and by multiple two-tailed unpaired t-test in e-g. In b, significant differences between genotypes are marked in the legend and differences with 4m WT in the graph. In e-g, significant differences between genotypes are marked in the graph and ANOVA indicates p value for time differences. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. ns = not significant. Numerical source data, statistics and exact p values and unprocessed blots are available as source data.

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Extended Data Fig. 8 Cell-type and tissue-specific changes in hepatic CMA activity during the light/dark cycle in different liver cells.

a. Immunofluorescence for Dendra and mac2 (to label hepatic Kupffer cells) in liver sections from KFERQ-Dendra mice at the indicated circadian times (CT). Nuclei are highlighted with Hoechst. Individual and merged channels are shown. Insets: higher magnification images. Dotted white line: Kupffer cells profile. b. Quantification of Dendra+ puncta per mac2+ cell section. n=3 mice per CT (50 cells were quantified per mouse and the mean value of puncta per cell in each mouse was used for statistics). c. Quantification of temporal changes in Dendra+ puncta number in hepatocytes and Kupffer cells relative to CT05 values (arbitrary value of 1). n=3 mice per CT (65 cells of each type were quantified per mouse and the mean value of puncta per cell in each mouse was used for statistics). d. Immunoblot of homogenate from control (CTR) and BMAL1 knockout (iBKO) mice livers at two circadian times (CT). Ponceau staining is shown as loading control. Right: Quantification of BMAL1 and HSC70, n=3 mice per CT and genotype. e,f. Immunoblot of lysosomes active (+) or inactive (-) for CMA isolated from livers of CTR and iBKO mice (e). Ponceau staining is shown as loading control. Quantification of immunoblots as the ones shown in e (f). n = 6 (CTR) and 3 (iBKO) mice per CT. g. Immunoblot for the indicated proteins in homogenate from kidneys of control (CTR) and BMAL1 knockout (iBKO) mice at two circadian times (CT). Three mice per condition are shown. Ponceau staining is shown as loading control. All values are mean+s.e.m. One-way ANOVA test followed by Tukey post-hoc tests (b) or two-way ANOVA test followed by Bonferroni’s (c) or Tukey’s (d,f) (post-hoc tests for multiple variable comparisons were used. Significant differences between genotypes are shown in the legend and between times in the graph in d. Significant differences between CMA+ and CMA- lysosomes for each genotype are marked in the graph in f and between lysosomes, times and genotype are summarized in the raw data file. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. ns = not significant. Numerical source data, statistics and exact p values and unprocessed blots are available as source data.

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Extended Data Fig. 9 Changes in the cyclic degradation of macroautophagy components upon CMA blockage.

Comparative differential proteomics of lysosomes from livers of wild-type (WT) or LAMP2A knockout mice (L2AKO) injected or not with leupeptin (Leu) and isolated at circadian time CT05 (day) or CT17 (night). All values are from n=3 mice per CT, genotype and treatment group. a. Principal component analysis showing the multivariate variation among the different genotype, time, and treatment groups. b. Percentage and number of proteins undergoing or not lysosomal degradation (increase upon leupeptin treatment). c. Preferences in degradation time of the subset of proteins degraded in lysosomes from L2AKO mice. d. Percentage of proteins in lysosomes from L2AKO mice displaying changes in their rate (magnitude) or time (timing) of degradation. e. Number of proteins in lysosomes according to their preference in degradation time and their dependence on the presence of LAMP2A in lysosomes. f,g. Lysosomal degradation of macroautophagy proteins LC3 (f) and p62 (g) calculated from the proteomic data. Graphs show quantification of levels of each of the proteins in lysosomes isolated from mice untreated (-) or injected with leupeptin (+L) (left) and lysosomal flux for both proteins calculated as differences in their lysosomal abundance between untreated or leupeptin injected mice (right). n=3 mice per CT, genotype and treatment with technical replicates for each one. Individual values per mouse (f,g) and mean+s.e.m are shown. Two-way ANOVA test followed by Bonferroni’s (f, g) post-hoc test (for multiple variable comparisons) was used. Differences were significant for ***p<0.001 and ****p<0.0001. ns = not significant. Numerical source data, statistics and exact p values are available as source data.

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Extended Data Fig. 10 Changes in proteasome components upon CMA blockage.

a, b. Chymotrypsin-like (CTL) and caspase-like (CSP) activities of the 20S and 26S proteasome in livers from WT or L2AKO mice. Time-course kinetics (a) and area under the curve (b) are shown. n=3 mice per genotype in a and b. c-g. Lysosomal degradation of proteasome subunits in the same mice. c shows heatmaps of degradation (green) or not (black) at the two time points and d-g shows changes in lysosomal levels of the indicated proteasome proteins as representative examples of the 4 identified patterns: subunits with comparable degradation and cycling in WT and L2AKO mice (d), subunits preferentially degraded during the day (e) or during the night (f) in WT mice that become continuously degraded in L2AKO mice, and subunits normally not degraded in lysosomes that become now lysosomal substrates (g). n=3 mice per CT, genotype and treatment with technical replicates for each one. Individual values per mouse and mean+s.e.m are shown. Two-way ANOVA test followed by Bonferroni’s (a) or Sidak’s (d-g) post-hoc tests (for multiple variable comparisons), and two-tailed unpaired t-test (b) were used. Differences were significant for *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. ns = not significant. Numerical source data, statistics and exact p values are available as source data.

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Supplementary information

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Supplementary Tables 1–10

Supplementary Table 1. Proteins degraded in lysosomes only in the early part of the circadian cycle (CT05). Logarithm of fold change (log2FC) in protein levels upon injection of leupeptin and P values calculated using unpaired two-tailed t-test. Supplementary Table 2. Proteins degraded in lysosomes only in the late part of the circadian cycle (CT17). Logarithm of fold change (log2FC) in protein levels upon injection of leupeptin and P values calculated using unpaired two-tailed t-test. Supplementary Table 3. Proteins degraded in lysosomes both in the early and late part of the circadian cycle. Logarithm of fold change (log2FC) in protein levels upon injection of leupeptin and P values calculated using unpaired two-tailed t-test. Supplementary Table 4. Lysosome-resident proteins which display circadian cyclic changes in their lysosomal abundance. Logarithm of fold change (log2FC) in lysosomal levels during day (D) and night (N) for proteins that do not undergo degradation in lysosomes and P values calculated using unpaired two-tailed t-test. Supplementary Table 5. Proteins no longer degraded in lysosomes from LAMP2A knock-out mice. Logarithm of fold change (log2FC) in protein levels upon injection of leupeptin and P values calculated using unpaired two-tailed t-test. Supplementary Table 6. Proteins degraded in a LAMP2A-dependent manner during the early or late part of the circadian cycle. Logarithm of fold change (log2FC) in protein levels upon injection of leupeptin and P values calculated using unpaired two-tailed t-test. Supplementary Table 7. Proteins that lose cyclic degradation in LAMP2A knock-out mice. Logarithm of fold change (log2FC) in protein levels upon injection of leupeptin and P values calculated using unpaired two-tailed t-test. Supplementary Table 8. Proteins that become lysosomal substrates only in LAMP2A knock-out mice. Logarithm of fold change (log2FC) in protein levels upon injection of leupeptin and P values calculated using unpaired two-tailed t-test. Supplementary Table 9. Antibodies used in this work, working dilution, company and catalogue number. Supplementary Table 10. Sequence of forward (F) and reverse (R) primers used for qPCR.

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Juste, Y.R., Kaushik, S., Bourdenx, M. et al. Reciprocal regulation of chaperone-mediated autophagy and the circadian clock. Nat Cell Biol 23, 1255–1270 (2021). https://doi.org/10.1038/s41556-021-00800-z

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