ER proteins decipher the tubulin code to regulate organelle distribution

Abstract

Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions^1,2. However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm³, forming abundant contacts with other organelles⁴. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer^5,6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180-microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.

Fig. 1. CLIMP63, p180 and KTN1 differentially regulate ER morphology.

a, Representative images of wild-type (WT), knockout (KO), double knockout (DKO) or triple knockout (TKO) of CLIMP63, p180 and/or KTN1 in U2OS cells stably expressing mEmerald–Sec61β (green, ER marker) and co-labelled with DAPI (blue, nuclear marker), anti-α-tubulin (red, microtubule marker) and anti-TRAPα (magenta, rough ER marker). Perinuclear and peripheral regions (left, outlined) are enlarged on the right. Scale bar, 10 μm. b, c, ER MDR (b) and asymmetry (c) (see Supplementary Text) in cells as in a. n = 30 cells. d–f, Quantifications of ER morphology in wild-type or CLIMP63-knockout (d), p180-knockout (e) or KTN1-knockout (f) cells expressing the indicated CLIMP63, p180 or KTN1 constructs. Since recombinant KTN1 levels are typically very low, only cells with detectable KTN1–mApple signals were quantified. Ability (+) or inability (−) of the mutants to bind microtubules (MT) is indicated. n = 32, 30, 30, 30, 31, 30 and 30 cells (left to right) in d; n = 59, 62, 64, 61, 34, 70 and 70 (left to right) in e; and n = 32, 38, 38, 39, 40 and 36 (left to right) in f. Data are mean ± s.d. with individual data points shown. Two-tailed t-test; P values are shown. Source data

Fig. 2. p180 and KTN1 bind glutamylated and polyglutamylated microtubules, respectively.

a, Representative micrographs of p180 and KTN1 fragments (cyan) binding to unmodified microtubules or microtubules glutamylated in vitro by TTLL6 (magenta). Internal reflection microscopy images for the microtubule channel were background subtracted and inverted. Average numbers of glutamate molecules (E) added to microtubules as quantified from mass spectroscopy data in each group are indicated. Scale bar, 5 μm. b, Binding of p180 and KTN1 fragments to unmodified and TTLL6-glutamylated microtubules. n = 110 (unmodified), 165 (α + 3.5 E, β + 1.3 E) and 112 (α + 8.3 E, β + 2.6 E) microtubules for p180; n = 141 (unmodified), 186 (α + 3.5 E, β + 1.3 E) and 156 (α + 8.3 E, β + 2.6 E) microtubules for KTN1. c, Representative micrographs of p180 and KTN1 microtubule interacting fragments (cyan) showing binding to unmodified microtubules or microtubules glutamylated by TTLL4 or TTLL7 (magenta). KTN1 and p180 are shown with different brightness/contrast settings for TTLL4- and TTLL7-modified microtubules, reflecting large differences in binding between mono- and polyglutamylated microtubules. Scale bar, 5 μm. d, e, Affinities of p180 and KTN1 for unmodified microtubules and microtubules glutamylated by TTLL7 (d) or TTLL 4 (e). The x-axis shows weighted averages of glutamate residues attached to α- and β-tubulin. n = 185 (unmodified) and 117 (α + 0 E, β + 1.2 E) microtubules for p180; n = 128 (unmodified) and 225 (α + 0 E, β + 1.2 E) microtubules for KTN1; n = 179 (unmodified) and 237 (α + 0 E, β + 1.5 E) microtubules for p180; n = 163 (unmodified) and 224 (α + 0 E, β + 1.5 E) microtubules for KTN1. f, CLIMP63 binds centrosomal microtubules, KTN1 binds perinuclear polyglutamylated microtubules, and p180 can bind peripheral microtubules with less glutamylation. Together, these proteins maintain proper asymmetric ER distribution, which regulates organelle distributions. MT, microtubules. Data are mean ± s.d. with individual data points shown. Kruskal–Wallis (b) or Mann–Whitney tests (d, e); P values are shown. Source data

Fig. 3. ER distribution changes during autophagy.

a, U2OS cells stably expressing mEmerald–Sec61β (green, ER marker) were starved in EBSS for 0, 0.5, or 2 h and labelled with anti-LAMP1 (red, lysosome marker). Scale bar, 10 μm. b, ER and lysosome distribution in wild-type, CLIMP63- or p180-knockout cells starved in EBSS for the indicated times. n = 55, 53, 52, 52, 53, 54, 51, 50, 51, 50, 46 and 44 cells (left to right) for both ER and lysosomes. c, Top, U2OS cells were starved in EBSS for 0–2 h and immunoblotted. Bottom, protein levels relative to α-tubulin. n = 5 experiments. d, Average intensities of PLA for p180 and KTN1 with α-tubulin at 0, 0.5, or 2 h of EBSS starvation or puromycin treatment (2 μg ml⁻¹ for 2 h). n = 273, 254, 188, 180, 143,144, 182 cells (left to right). e, Schematic of p180 domain composition. The ribosome-binding domain includes 41 positively charged decapeptide repeats, which can potentially bind microtubules once ribosomes are dissociated. Amino acid sequences for several repeats are shown, with positively charged residues in red and negatively charged residues in green. Repeat sequences vary slightly but are all positively charged. f, Under normal cellular conditions, p180L ribosome-binding repeats are occupied by ribosomes and cannot bind microtubules. When cells are starved, ribosomes dissociate from ER, and the repeats can then bind microtubules. g, PLA for p180 and RPL3 (ribosome marker) upon starvation or puromycin treatment (2 μg ml⁻¹ for 2 h). n = 230 cells. Data are mean ± s.d. with individual data points shown. One-way ANOVA followed by Dunnett’s multiple comparisons test for (b), (c); two-sided t-test for (d), (g); P values are shown. See Supplementary Information for uncropped western blots. Source data

Extended Data Fig. 1. Knockout of CLIMP63, p180 and KTN1, and resulting ER phenotypes.

a, Schematic illustration of CLIMP63, p180 and KTN1 protein domains. Purple numbers indicate key amino acids. Shorter isoform of p180 (p180s, Uniprot Q9P2E9.5) is also shown. b, Western blotting (WB) of the indicated wild-type (WT) or knockout (KO) cells. The lower band in the KTN1 blots (indicated with an asterisk) corresponds to the shorter cytosolic isoform of KTN1. See Supplementary Information for uncropped western blots. c, Representative images of three patterns of ER distribution in U2OS cells. “Dispersed” (left) is characterized by dominant sheets or matrices at the cell periphery; “Clustered” (right) is characterized by asymmetric dense accumulation of perinuclear ER at one side of the nucleus; all other ER types are considered “Perinuclear”. d, Proportion of wild-type or indicated KO cells with different patterns of ER distribution. n = 3 experiments with at least 200 cells counted in each experiment. e, ER distribution of wild-type or CLIMP63, p180 or KTN1 KO cells treated with 5 μM etoposide or 100 nM camptothecin for 24 h to synchronize cells in S/G2 phase. n = 3 experiments with at least 200 cells counted in each experiment. f, ER distributions in wild-type or CLIMP63, p180 or KTN1 KO cells treated with 10 μM nocodazole for 24 h and released for 6 h to synchronize cells in G1 phase. n = 3 experiments with at least 200 cells counted in each experiment. g, Representative images of perinuclear ER in wild-type or CLIMP63, p180 and KTN1 triple-KO cells, showing LUT color grading according to intensity of the ER marker mEmerald-Sec61β. Scale bars, 10 μm. All bars represent mean ± s.d. Source data

Extended Data Fig. 2. Methods for quantifying organelle distribution, and quantifications of TRAPα and microtubule distribution in knockout cells.

a, Summed projections generated from three-dimensional Airyscan images (left). Fluorescence from neighboring cells is removed and the center of the nucleus is manually selected to function as the origin (yellow dot in right image). Fluorescence intensities are converted to probabilities (right image, see Supplementary Text). Scale bar, 10 μm. b, A radius is drawn out from the center of the nucleus past the farthest point on the cell and swept through 360° in 0.1° intervals, taking a line profile each time. The nuclear envelope and edge of the cell are identified at each radius. c, Resulting probabilities of each channel in r- and θ-space, represented as fluorescence intensities. Red and yellow dashed lines indicate the approximate location of the nuclear envelope and the cell edge, respectively (left panel), as shown in (b). The probability distributions of nuclear and ER signals are normalized to correct for cell shape (right panel). Dashed lines indicate the location of the nuclear envelope and cell edge after normalization. d, Associated radial distributions of probabilities as measured in terms of distance across the cytoplasm as in (c). Relative probability (y-axis) indicates any single molecule of DAPI (nucleus) or mEmerald-Sec61β (ER) falling at a specific proportion of the distance between the nuclear envelope and the edge of the cell (x-axis). ER MDR represents the average distance of the ER on this scale and can be used to quantify the propensity of the ER to penetrate the cellular periphery; higher MDRs indicate a larger proportion of the ER in the periphery. e–g, A radius is drawn out past the farthest point on both sides of the cell and swept through 180° in 0.1° intervals, taking a line profile each time (e). The edge of the cell is identified at each radius. Resulting intensity distribution across all radii, with the red line indicating the center of the nucleus (f). For each radius, the difference between two sides of the center (ΔF) is calculated and plotted as a function of θ. The asymmetry value is then calculated as a sum of the exact values of ΔF (g). h, i, Quantifications of TRAPα (rough ER) distributions in WT or the indicated KO cells. n = 31 cells. j, k, Quantifications of microtubule (labeled with anti-α-tubulin) distribution for wild-type or the indicated KO cells. n = 41, 19, 25, 21, 26, 24, 46, 25, 27, 24, 23, 30, 25, 30, 31 cells (left to right) for j; n = 22, 22, 30, 30, 30, 27, 31, 30, 30, 23, 26, 29, 24, 30, 31 cells for k. l, m, Quantifications of ER and microtubule MDR for more cells to show differences in microtubule MDR. n = 210 cells for l, n = 197, 161, 162 cells for m. All bars represent mean ± s.d. P values are shown on top; differences without labeling are not significant, comparisons are with the wild-type group using two-tailed t-tests. Source data

Extended Data Fig. 3. Microtubule sedimentation assays of ER proteins.

a, Microtubule co-sedimentation assays of U2OS cells. The pellet (P) of 37 °C incubation indicates the microtubule-bound fraction, and supernatant (S) indicates the unbound fraction. 4 °C incubation acts as a microtubule-free control. b, Microtubule co-sedimentation assays of U2OS cells with exogenous CLIMP63-HA, p180s-myc or KTN1-myc expression. Proteins were expressed in corresponding knockout cells. Note that only one representative α-tubulin blot (from the CLIMP63 assay) is shown. c, p180 is very unstable after cell lysis. The input sample was collected by directly adding sample buffer (50 mM Tris, pH 6.8, 1 mM DTT, 10% glycerol, 2% SDS, 0.1% Bromophenol Blue) onto the plate followed by immediate boiling. Other samples were incubated in lysis buffer (50 mM Tris, pH7.4, 150 mM NaCl, 1% Triton X-100, 1 mM DTT, and protease inhibitor cocktail) at room temperature or on ice for the indicated times before adding sample buffer and boiling. d, Western blotting of WT or p180 knockout (KO) U2OS or COS7 cells, showing that only the long isoform is detectable in these cell lines. e, Detailed mapping of microtubule-binding domains of CLIMP63. f, Mapping of microtubule-binding domains of p180. Amino acid sequences around key microtubule-binding sites are shown at the bottom. Note that this part of the sequence is present in both long and short isoforms of p180. Positively charged amino acids are shown in red. Segments (amino acids 51-80) necessary for microtubule binding are underlined. g, Mapping of microtubule-binding domains of KTN1. Amino acid sequences around key microtubule-binding sites are shown at the bottom. Positively charged amino acids are in red. Segments (amino acids 112-120) necessary for microtubule binding are underlined. See Supplementary Information for uncropped western blots.

Extended Data Fig. 4. Rescue assays.

a, Western blots of wild-type or CLIMP63 knockout cells transfected with empty vector or the indicated CLIMP63 constructs. b, Representative fluorescence images and quantifications of CLIMP63 knockout cells transfected with empty vector or the indicated CLIMP63 constructs. c, Western blots of wild-type or CLIMP63 knockout cells transfected with empty vector or the indicated CLIMP63 constructs. CLIMP63-3SE indicates S3E, S17E and S19E triple mutation of CLIMP63. d, Quantifications of ER morphology in wild-type or CLIMP63 KO cells expressing the indicated CLIMP63 constructs. n = 43, 31, 46 cells (left to right). e, f, Western blots and representative images of wild-type or p180 KO cells expressing the indicated p180 constructs. g–i, Western blots and representative images of wild-type or KTN1 KO cells expressing the indicated KTN1 constructs. j–l, p180 knockout U2OS cells were transfected with mApple, p180s-mApple, or p180s mutants lacking kinesin-binding domain (KBD), then imaged and quantified for ER morphology. Scale bars, 10 μm. n = 82, 53, 53 cells for both (k) and (l) (left to right). Data are mean ± s.d. P values shown on top, two-tailed t-tests. See Supplementary Information for uncropped western blots. Source data

Extended Data Fig. 5. PLA of CLIMP63, p180 and KTN1 with α-tubulin, and depleting centrosome or Golgi-derived microtubules affects ER distribution.

a, N-terminal mEmerald tag was knocked into endogenous CLIMP63 using CRISPR/Cas9 to facilitate PLA, as no anti-CLIMP63 antibody targeting its cytosolic region was available. Western blots of wild-type or knock-in cells are shown. b, Representative PLA images of p180 or KTN1 with α-tubulin. Note that all PLA dots (a dot indicates one binding event) are localized adjacent to both ER and microtubules. c, Representative PLA images of endogenous mEmerald-CLIMP63 with α-tubulin. d, Western blot analysis of cells transfected with control or AKAP450 siRNAs. e–h, Representative data and quantifications for PLA of CLIMP63, p180, or KTN1 with α-tubulin, indicative of microtubule binding. Cells are either untreated, treated with centrinone B (CNB) to deplete the centrosome, or transfected with control siRNA or siAKAP450 (to deplete Golgi-derived microtubules). n = 130, 134, 142, 143 cells (left to right) for (f), n = 116, 139, 140, 150 cells for (g), n = 119, 117, 139, 166 cells for (h). i, j, Asymmetry and MDR quantifications for PLA signals between CLIMP63, KTN1, or p180 with α-tubulin. n = 83, 74, 72, 40, 38 cells (left to right) for (i), n = 81, 79, 82, 43, 44 cells for (j). k, Representative images of U2OS cells with or without CNB treatment. Cells stably expressing mEmerald-Sec61β (green, ER) are stained with DAPI (blue, DNA) and immunolabeled with anti-pericentrin (red) and anti-α-tubulin (magenta) antibodies. Perinuclear and peripheral regions in the cells (boxed) are enlarged at the right. l, Quantifications of ER MDR in wild-type or p180 knockout (KO1 and KO2) cells, with or without CNB treatment. n = 31, 30, 31, 43, 31, 39 cells (left to right). m, n, Representative images and quantifications of ER asymmetry for wild-type or CLIMP63/KTN1 double-knockout cells. Cells were transfected with control siRNA or AKAP450 siRNA to deplete Golgi-derived microtubules. n = 31, 31, 31, 32, 31, 32 cells (left to right). Scale bars, 10 μm. Data are mean ± s.d. P values shown on top, two-tailed t-tests. See Supplementary Information for uncropped western blots. Source data

Extended Data Fig. 6. Microtubule binding of CLIMP63, p180 and KTN1 are differently affected by microtubule glutamylation.

a, Western blots of wild-type or the indicated knockout U2OS cells. b, c, Western blots and quantifications of U2OS cells either treated with CNB to deplete the centrosome or else transfected with control siRNA or siAKAP450 (to deplete Golgi-derived microtubules). n = 6 experiments. d, Representative images of U2OS cells transfected with CLIMP63-mEmerald, p180-mEmerald, or KTN1-mEmerald, with or without co-expression of TTLL7. Note that CLIMP63 overexpression leads to dramatic ER-microtubule alignment (~80% of cells), while p180 and KTN1 require co-expression of TTLL7 (which polyglutamylates microtubules) for robust ER-microtubule alignment. Scale bar, 10 μm. e, Quantifications of microtubule-ER alignments in cells transfected with the indicated expression plasmids; n = 4 experiments, with at least 100 cells counted in each experiment. f, Quantifications of microtubule-ER alignments in cells treated with or without CNB, and overexpressing CLIMP63-mEmerald. n = 4 experiments, with 100 cells counted per experiment. g, Schematic diagram depicting how microtubule glutamylation levels are modulated by actions of tubulin glutamylases (TTLLs) and deglutamylases (CCPs). h, Western blots of U2OS cells overexpressing TTLL4 or TTLL7. polyE detects microtubule polyglutamylation (at least 2 glutamates in the side chain); GT335 reacts with branch points of microtubule glutamylation and thus detects both mono- and polyglutamylation. i, Western blots of U2OS cells overexpressing CCP1 or CCP5. Cells were subjected to microtubule sedimentation, and pellets (P, microtubule fraction) and supernatants (S, soluble fraction) at 37 °C were analyzed. j–l, Relative PLA intensities for CLIMP63, p180, or KTN1 with α-tubulin (indicative of microtubule binding) in U2OS cells overexpressing the indicated TTLLs or CCPs. n = 138, 103, 112, 99, 102 cells (left to right) for (j); n = 112, 113, 107, 131, 121 cells for (k); n = 188, 108, 113, 123, 109 cells for (l). m, Ponceau S staining of p180 and KTN1 proteins purified from HEK293T cells. n, Coomassie Brilliant Blue staining of purified CLIMP63 from HEK293T cells. o, p, Mass spectra of microtubules glutamylated by TTLL6 for different times, or by else TTLL4 or TTLL7, and used for in vitro microtubule-binding assays. Spectra display characteristic distributions of masses with peaks separated by 129 Da (corresponding to one glutamate). Peak labels show number of glutamates on α- and β-tubulin. Glutamate numbers are indicated in green, dark green, orange, and red for α1B, α1C, βI and βIVb isoforms, respectively. q, Representative micrographs of CLIMP63-mNeonGreen-2×Strep (CLIMP63, cyan) showing binding to unmodified microtubules or else microtubules (magenta) glutamylated in vitro by TTLL7. Interference reflection microscopy images for the microtubule channel were background subtracted and inverted. Average numbers of glutamates added to microtubules as quantified from mass spectrometry data for each group are shown on top. Scale bar, 5 μm. r, Affinities of CLIMP63 for unmodified microtubules and microtubules glutamylated by TTLL7. X-axis indicates the weighted average of glutamate residues attached to α- and β-tubulin. n =107, 61, 127, and 136 microtubules with unmodified, α+0E/β+1.4E, α+0E/β+2.7E, and α+0.1E/β+3.8E microtubules, respectively. Data are mean ± s.d. P values shown on top, Mann-Whitney test for panel r, two-tailed t-tests for others. See Supplementary Information for uncropped western blots. Source data

Extended Data Fig. 7. Modulating tubulin glutamylation affects ER distribution.

a, Representative images of U2OS and COS7 cells sequentially immunolabeled for polyE (polyglutamylation), polyE plus GT335 (glutamylation) and Alexa 647 conjugated α-tubulin. GT335 signal indicates monoglutamylation in this case because polyglutamylation is pre-saturated with polyE antibody. b, Quantifications of signal distributions for cells as in (a). Data points from the same cell are linked by solid lines. n = 20 cells for U2OS, 35 cells for COS7. c, Western blot analysis of U2OS, COS7, HeLa and RPE1 cells. d, MDR ratios of monoE (indicated by GT335 labeling) to polyE, or microtubules (indicated by α-tubulin labeling) to polyE. n = 20 cells for U2OS, 35 cells for COS7. e, Representative images of U2OS cells overexpressing the indicated TTLLs or CCPs. f, g, Quantifications of ER and microtubule MDRs of cells as in e; n = 37, 40, 34, 40, 40, 41, 39 cells (left to right) for (f); n = 29, 38, 37, 51, 38, 46, 39 cells for (g). h, Quantifications of ER MDR of cells in p180/KTN1 double knockout cells overexpressing the indicated TTLLs or CCPs; n = 63, 61, 64, 63, 61 cells (left to right). i, Quantifications of ER MDR of wild-type or KTN1 knockout cells overexpressing TTLL7; n = 40, 64 cells (left to right). j, U2OS cells transfected with the indicated siRNAs for 72 h were analyzed by western blotting. k, U2OS cells transfected with the indicated siRNAs for 48 h were analyzed by real time PCR. n = 4 repeats. l, m, Representative image and quantification of ER in control or CCP5 siRNA transfected cells. Scale bars, 10 μm. n = 64, 70, 69, 39, 39, 39 cells (left to right). Data are mean ± s.d. P values shown on top, two-tailed t-test. See Supplementary Information for uncropped western blots. Source data

Extended Data Fig. 8. Knockout of CLIMP63, p180 and KTN1, or overexpression of CCPs in COS7 cells changes ER distribution.

a, Western blots of wild-type or else CLIMP63, p180, or KTN1 knockout cells. b, Representative images of wild-type or CLIMP63, p180, KTN1 knockout cells. ER is labeled by endogenous mEmerald-calreticulin. Scale bar, 10 μm. c, d, Quantifications of ER distribution. n = 60, 60, 58, 96, 88, 96, 51 cells (left to right) for both (c) and (d). e–h, Wild-type or p180 knockout COS7 cells with endogenous mEmerald-calreticulin (ER, green) were transfected with CCP5-mApple or CCP6-mApple for 24 h and analyzed by western blotting (e) and confocal imaging (f). ER distributions are quantified (g, h). Scale bar, 10 μm. n = 39, 49, 45, 41, 40 cells (left to right) for both (g) and (h). Data are mean ± s.d. P values shown on top, two-tailed t-tests. See Supplementary Information for uncropped western blots. Source data

Extended Data Fig. 9. Organelle distribution in knockout cells.

a, Simultaneous live imaging of six organelles with spectral unmixing. Two representative cells are shown. Note that where there is more ER, there tends to be more of the other organelles. b, Representative images of organelle distributions in CLIMP63 or p180 knockout cells. Markers used: mEmerald-Sec61β for ER; anti-TOM20 for mitochondria; anti-GM130 for Golgi apparatus; anti-EEA1 for endosomes; anti-LC3 for autophagosomes; anti-catalase for peroxisomes, anti-Lamp1 for lysosomes; and LD540 for lipid droplets. Cells labeled for autophagosome distribution were starved in EBSS for 2 h. ER is shown in green, while other organelles are in red. c, d, Quantifications of the distributions of different organelles labeled with specific markers in wild-type or else CLIMP63, KTN1, or p180 knockout cells. n = 30, 31, 30, 46 cells (left to right) for mitochondria MDR, n = 83, 86, 87, 46 cells for lipid droplet MDR, n = 30, 31, 31, 46 cells for peroxisome MDR, n = 29 cells for Golgi MDR, n = 30 cells for Autophagosome and lysosome MDR; n = 51, 46 cells for mitochondria asymmetry, n = 29, 31 cells for Golgi asymmetry, n = 49, 50 cells for lipid droplet asymmetry, n = 31 cells for autophagosome, lysosome and peroxisome asymmetry. e–g, Quantifications of lysosome, mitochondria, and peroxisome distributions in wild-type or p180/KTN1 double knockout cells transfected with control vector or CCP1. n = 32, 36, 36, 37 cells (left to right) for (e) and (f), n = 41, 48, 44, 40 cells for (g). Scale bars, 10 μm. All bars represent mean ± s.d. with individual data points shown. P values are shown along the top, two-tailed t-tests. Source data

Extended Data Fig. 10. CLIMP63 and p180 regulate autophagic flux.

a, U2OS cells were pre-treated with the indicated compounds for 2 h (except etoposide, which was added for 16 h), then with EBSS plus the same compound for 30 min, and then subjected to western blotting. Compounds used: 1 μM lysosome degradation inhibitor DC661, 1 μM proteosome degradation inhibitor MG132, 6 μg/mL ER translation inhibitor puromycin, 100 μg/mL protein translation inhibitor cycloheximide (CHX), 50 μM etoposide that causes DNA damage and also blocks protein expression, and 1 μm N-linked glycosylation inhibitor tunicamycin. b, Western blot analysis of wild-type, CLIMP63 or p180 knockout U2OS cells (Parental, no stable mEmerald-Sec61β expression). c, Representative images and quantifications for wild-type, CLIMP63 or p180 knockout U2OS cells expressing GFP-mCherry-LC3. The GFP signal is quenched by the acidic environment of lysosomes, so only autophagosomes that have not yet fused with lysosomes have green GFP signal, while autolysosomes only exhibit mCherry signal. n = 40 cells. Scale bar, 10 μm. d, Wild-type or CLIMP63 knockout cells were starved in EBSS for 0 or 8 h, with or without brefeldin A treatment to block lysosomal degradation, and then immunoblotted. Relative amounts of autophagic substrate p62 are shown in the lower panel. n = 4 experiments. e, Cathepsin L activity as determined by substrate reaction. n = 4 experiments. f, Lysosome acidification analysis using Lysosensor Green. n = 200 cells. g, Wild-type or p180 knockout cells were starved in EBSS for 0 or 8 h, with or without brefeldin A treatment to block lysosomal degradation, and western blotted. h, Wild-type or p180 knockout cells were starved in EBSS for 2 h, then re-supplemented with regular medium for 0-30 min. Cells were immunoblotted for phosphorylated S6K (p-S6K) and total S6K to indicate activity of mTOR signaling. n = 4 experiments. i, Western blots reveal the indicated microtubule modifications with or without EBSS starvation for 0.5 and 2 h. Relative intensities of GT335 and polyE immunoreactive signals are quantified at the right. n = 5 experiments. j, Representative images for PLA of p180 and the ribosomal marker RPL3 at 0 or 2 h of EBSS starvation, or else for cells treated with 2 μg/mL puromycin for 2 h. Scale bar, 50 μm. k, Representative images of U2OS cells transfected with mEmerald-Sec61β, p180s-mEmerald, or p180L-mEmerald with or without co-transfection of TTLL7-3×flag. Scale bar, 10 μm. l, Quantifications of microtubule alignments in cells transfected with the indicated constructs; n = 4 experiments, with at least 100 cells counted per experiment. m, Microtubule alignments of p180L-mEmerald in cells treated with EBSS or 2 μg/mL puromycin for 2 h. n = 6 experiments, with at least 100 cells counted per experiment. Data are mean ± s.d. P values are shown along the top, two-tailed t-tests. See Supplementary Information for uncropped western blots. Source data