Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment1,2,3. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system4,5,6, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease)7. ER-to-Golgi trafficking of CLN8 requires interaction with the COPII and COPI machineries via specific export and retrieval signals localized in the cytosolic carboxy terminus of CLN8. CLN8 deficiency leads to depletion of soluble enzymes in the lysosome, thus impairing lysosome biogenesis. Binding to lysosomal enzymes requires the second luminal loop of CLN8 and is abolished by some disease-causing mutations within this region. Our data establish an unanticipated example of an ER receptor serving the biogenesis of an organelle and indicate that impaired transport of lysosomal enzymes underlies Batten disease caused by mutations in CLN8.

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Data availability

The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary Information files. Source data for Figs. 1–5 and Supplementary Figs. 2–5 have been provided as Supplementary Table 1. All other data supporting the findings of this study are available from the corresponding author upon request. Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD011066. The GEO accession numbers of the microarray data sets analysed for the co-expression analysis are the following: GDS1237, GDS1249, GDS1344, GDS1369, GDS1411, GDS1413, GDS1427, GDS1439, GDS1553, GDS1579, GDS1580, GDS1604, GDS1617, GDS1665, GDS1667, GDS1673, GDS1685, GDS1732, GDS1779, GDS1807, GDS1812, GDS1869, GDS1917, GDS1962, GDS1973, GDS1989, GDS2010, GDS2023, GDS2046, GDS2052, GDS2083, GDS2088, GDS2089, GDS2118, GDS2125, GDS2154, GDS2164, GDS2189, GDS2204, GDS2213, GDS2215, GDS2216, GDS2221, GDS2250, GDS2251, GDS2307, GDS2339, GDS2374, GDS2414, GDS2416, GDS2418, GDS2426, GDS2431, GDS2432, GDS2453, GDS2470, GDS2471, GDS2484, GDS2486, GDS2491, GDS2495, GDS2499, GDS2526, GDS2534, GDS2548, GDS2565, GDS2604, GDS2609, GDS2611, GDS2615, GDS2628, GDS2635, GDS2653, GDS2657, GDS2697, GDS2724, GDS2728, GDS2737, GDS2749, GDS2750, GDS2755, GDS2760, GDS2772, GDS2779, GDS2782, GDS2789, GDS2794, GDS2819, GDS2821, GDS2822, GDS2832, GDS2835, GDS2838, GDS2860, GDS2902, GDS2919, GDS2935, GDS2958, GDS2959, GDS3062, GDS3217, GDS3220, GDS3223 and GDS651.

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We thank P. Lobel, D. Pearce, J. Cooper, T. Dierks, M. Damme, Z. Liu and S. Elsea for helpful discussion; A. Schiano and M. Rousseaux for technical assistance; H. Zoghbi, V. Brandt, A. Ballabio, H. Jafar-Nejad, K. Venkatachalam and M. Wang for critical reading of the manuscript; and B. Turk (J. Stefan Institute, Slovenia), K. Yamada (Institute for Developmental Research, Japan), B. Blazar (University of Minnesota, USA), D. Kohn (University of California, Los Angeles, USA), T. Beccari (Università degli Studi di Perugia, Italy) and M. Peterfy (University of California, Los Angeles, USA) for providing plasmids encoding lysosomal proteins or other tested proteins. This work was supported by the NIH grant NS079618 (to M.S.) and grants from the Beyond Batten Disease Foundation (to M.S.) and the NCL-Stiftung (to M.S.). This project was supported in part by the Hamill Foundation and by IDDRC grant number 1U54 HD083092 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

Author information


  1. Department of Molecular and Human Genetics, Baylor College of Medicine, Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX, USA

    • Alberto di Ronza
    • , Lakshya Bajaj
    • , Jaiprakash Sharma
    • , Deepthi Sanagasetti
    • , Parisa Lotfi
    • , Carolyn Joy Adamski
    • , Michela Palmieri
    • , Abdallah Amawi
    • , Kevin Tommy Chang
    •  & Marco Sardiello
  2. Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX, USA

    • John Collette
    •  & Richard Norman Sifers
  3. Departments of Bioengineering, Chemical and Biomolecular Engineering, and Biochemistry and Cell Biology, Rice University, Houston, TX, USA

    • Lauren Popp
    •  & Laura Segatori
  4. Department of Biosciences, Rice University, Houston, TX, USA

    • Kevin Tommy Chang
  5. Department of Neurological and Movement Sciences, University of Verona, Verona, Italy

    • Maria Chiara Meschini
    •  & Alessandro Simonati
  6. Departments of Medicine, Pediatrics, and Molecular and Cellular Biology, Dan Duncan Cancer Center, Baylor College of Medicine, Houston, TX, USA

    • Hon-Chiu Eastwood Leung
  7. IRCCS Stella Maris, Pisa, Italy

    • Filippo Maria Santorelli


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M.S. conceived and supervised the study. A.d.R. and M.S. designed the experiments and analysed the data with the contribution of R.N.S., L.S., A.S. and F.M.S. A.d.R. performed the confocal analysis, flow cytometry, the cycloheximide assay, qPCR and enzyme assays. A.d.R. and L.B. performed subcellular fractionation. A.d.R. and J.S. performed the Co-IP experiments. A.d.R., L.B., J.S., D.S., P.L., C.J.A., J.C., M.P., A.A., L.P., K.T.C. and M.C.M. performed cloning, cell culture and transfection experiments. A.d.R. and P.L. performed immunohistochemistry. H.-C.E.L. performed LC–MS/MS. M.S. performed the co-expression and evolutionary analyses. A.d.R. and M.S. wrote the manuscript with help from H.-C.E.L., L.S., A.S. and F.M.S. All authors reviewed and edited the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Marco Sardiello.

Integrated supplementary information

  1. Supplementary Figure 1 Expression and interaction analyses of candidate cargo receptors.

    (a), ER localization of YFP-tagged candidate cargo receptors. Confocal microscopy analysis of Y1- or Y2-tagged ER candidate proteins (green) shows co-localization of all of the constructs with the ER marker, KDEL (red), and no discernible alteration of ER morphology following protein overexpression. Scale bar: 20 μm. (b), Shown is a BiFC assay of candidate cargo receptors tagged with Y1 and five pools of Y2-tagged lysosomal enzymes, each pool containing 10 different enzymes. Control experiments (CTRL) were performed by co-transfecting Y1-tagged candidates with pools of Y1-tagged lysosomal enzymes. Scale bar: 200 μm. (c), Details of the BiFC-flow cytometry analysis. CLN8 proteins form homodimers and this property has been used to set up the BiFC-flow cytometry assay. Shown are examples of histogram representations of BiFC data for CLN8 Y1/Y1 and Y1/Y2 controls (left), CLN8/TPP1 interaction (center), and CLN8/PRCP lack of interaction (right). In all analyses, cells were co-transfected with a Ruby plasmid (red fluorescence), used to normalize BiFC readings for transfection efficiency. Images shown in (a), (b) and (c) are representative of n= 3 independent experiments

  2. Supplementary Figure 2 Analysis of lysosome-enriched fractions from WT and CLN8-deficient mice.

    (a), Lysosome-enriched fractions were harvested upon liver homogenization and ultra-centrifugation in a discontinuous Nycodenz gradient. (b), HEXA activity assay showed significant enrichment for lysosomes in the collected fractions and no obvious changes in activity in the comparison of WT and Cln8mnd mice. (c), LAMP1 immunoblot showing lysosomal enrichment in collected fractions and no obvious changes between WT and Cln8mnd samples. (d), Details of the plot of relative protein signals from LC-MS/MS analysis of lysosome-enriched liver fractions from WT and CLN8-deficient mice (KO). Each blue dot (representing soluble lysosomal enzymes) has been labeled with the corresponding protein symbol; red dots and grey dots represent lysosomal membrane proteins and other proteins detected by LC-MS/MS, respectively. Data are relative to n = 2 independent proteomic analyses conducted on pools of three livers each. (e), GSEA of lysosomal membrane proteins and mitochondrial matrix proteins in the comparison of lysosome-enriched fractions from the livers of WT and CLN8-deficient mice. Proteins detected by LC-MS/MS are ranked according to their enrichment in CLN8-deficient vs. WT mice (left: increased in CLN8-deficient mice, red; right: decreased in CLN8-deficient mice, blue). GSEA shows that the sets of proteins are not differentially distributed in CLN8-deficient vs. WT mice (Plysosomal membrane proteins = 0.31; Pmitochondrial matrix proteins = 0.32). (f), Enzymatic assay of GAA, GALNS, GBA, GLB1 and TPP1 in liver lysosome-enriched fractions from 2-month-old WT and Cln8mnd mice. Activity is expressed as percentage of the WT samples. All enzyme activities were conducted by using fluorophore analogs of enzymes substrates, as previously described: TPP11; GAA2; GBA3; GLB14; GALNS5. (g), Expression analysis of lysosomal genes in the liver of 2-month-old WT and Cln8mnd mice. Shown is the fold-change in expression of genes in the Cln8mnd relative to WT mice. Expression levels are normalized to the housekeeping gene Ppif (peptidylpropyl isomerase F or cyclophilin D). Images shown in (a) and (c) are representative of n = 3 independent experiments. In (b), (f) and (g), data are means ± SEM (n = 3 independent experiments, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test)

  3. Supplementary Figure 3 Depletion of lysosomal enzymes in tissues from CLN8-deficient mice.

    (a), Immunohistochemistry of lysosomal proteins in liver and brain from WT and CLN8-deficient mice. Liver and brains were collected from 2-month-old Cln8mnd and WT mice. CTSD and TPP1 protein levels are strongly reduced in CLN8-deficient mice, compared to WT, in all tested conditions. LAMP1 immunostaining is unchanged. Scale bar, 100 μm. (b), Confocal microscopy of lysosomal enzymes (red) and LAMP1 (green) on brain sections of WT and CLN8-deficient mice. Pearson correlation showed a significant reduction of the enzyme/LAMP1 signal overlap in CLN8-deficient mice compared to WT mice. Insets show four-fold magnification. Scale bar: 20 μm. Images in (a) are representative of n = 3 independent experiments. In (b), data are means ± SEM (n = 3 independent experiments, n = 10 independent images quantified, **P < 0.01, ****P < 0.0001, two-tailed Student’s t-test)

  4. Supplementary Figure 4 Analysis of lysosomal protein localization and CLN8 interactions.

    (a), Confocal microscopy of brain sections from WT and CLN8-deficient mice using antibodies against lysosomal enzymes (red) and the ER marker KDEL (green). Scale bar is 20 μm. (b), TPP1/LAMP1 overlap analysis of two CLN8 patients’ fibroblasts. Skin biopsied fibroblasts from two healthy subjects were used as controls. Data are means ± SEM (n = 3 independent experiments, n = 10 independent images quantified, ***P < 0.001, two-tailed Student’s t-test). (c), Immunoblot analysis of skin biopsied fibroblasts from two controls and from two CLN8 patients showed depletion for all the lysosomal protein tested. Signals are normalized to GAPDH staining. (d), Confocal microscopy showing colocalization of CLN8-myc (green) with the ER marker, KDEL (red). Scale bar is 20 μm. (e), Co-IP assay of γ-COP and CLN8 in the presence or absence of CBM. pcDNA is used as an empty vector control. Input represents 10% of the total cell extract. (f), Amino acid sequence of CLN8’s cytosolic C-terminus. Shown are the signals for COPI and COPII recognition and the changes in the CLN8dK and CLN8WdK constructs. TM, transmembrane domain. (g), BiFC analysis of CLN8dK and CLN8WdK constructs showing that CLN8 dimerization and CLN8 competency in the BiFC assay are not affected by mutagenesis of CLN8 ER retrieval and export signals. Scale bar is 200 μm. Images shown in (a), (c), (d), (e) and (g) are representative of n = 3 independent experiments

  5. Supplementary Figure 5 Generation of CLN8-/- cells and their use in protein maturation experiments.

    (a), Generation of CLN8-/- cells by CRISPR/Cas9 genome editing. The schematic shows the targeted region of CLN8 gene before and after the editing. (b), Real-time qPCR showing lack of expression of CLN8 in CLN8-/- cells. Fold changes are normalized to the housekeeping gene, GAPDH. (c), Metabolic radiolabeling in CLN8-/- cells and their parental HeLa cells, with or without CLN8 re-expression, showing rescue of the mature forms of CTSD and PPT1. CTSD and PPT1 were immunoprecipitated by using antibodies against the endogenous proteins. CLN8 was expressed by using a doxycycline-inducible vector used to transduce CLN8-/- and control cells. Arrows indicate the mature, lysosome-associated enzyme. IP = immature protein. ML = Mature light. (d), Immunoblot analysis of CTSD, GALNS, PPT1 and TPP1 in CLN8-/- cells and their parental HeLa cells treated with 1 µM MG132 and 1 µM Eeryastatin I (M + E) for 24 hours or left untreated. CTSD IP, immature precursor. CTSD ML, mature light form. GALNS I, immature form. GALNS M, mature form. An antibody against ubiquitin was used as a control for ERAD inhibition. (e), Metabolic radiolabeling of CTSD in CLN8-/- cells and their parental HeLa cells upon treatment with 10 µM MG132 and 6 µM Eeryastatin I (M + E), which were added at the beginning of the starvation step (1 hour) and kept in the pulse and chase media. The arrow indicates the mature, lysosome-associated enzyme. In (be), data are means ± SEM (n = 3 independent experiments, *P < 0.05, **P < 0.01, two-tailed Student’s t-test)

  6. Supplementary Figure 6

    Unprocessed images of all blots

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–6 and Supplementary Table legends.

  2. Reporting Summary

  3. Supplementary Table 1

    Statistics source data.

  4. Supplementary Table 2

    ER genes whose expression correlated with that of lysosomal genes.

  5. Supplementary Table 3

    GSEA of lysosomal soluble proteins.

  6. Supplementary Table 4

    GSEA of lysosomal membrane proteins.

  7. Supplementary Table 5

    GSEA of mitochondrial matrix proteins.

  8. Supplementary Table 6

    Oligonucleotide sequences.

  9. Supplementary Table 7

    List of primary and secondary antibodies.

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