Nature - USA (2020-02-13)

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nature research | reporting summary

October 2018

Corresponding author(s): Keiji Tanaka and Yasushi Saeki

Last updated by author(s):Nov 21, 2019

Reporting Summary

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Statistics

For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement

A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly

The statistical test(s) used AND whether they are one- or two-sided

Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of all covariates tested

A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons

A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient)

AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)

For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings

For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes

Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated

Our web collection on statistics for biologists contains articles on many of the points above.

Software and code

Policy information about availability of computer code

Data collection The images of the live cells (Fig. 1a, 2d, 3a; Extended Data Fig. 2b, 7b; Supplementary Video 1, 2, 3, 4, 5, 6) and the two-color or three-

color samples of the fixed cells (Fig. 1a, 1c, 2c, 3b, 4a, 4c; Extended Data Fig. 1a, 2a, 2c, 2e, 3a, 3b, 3c, 5a, 5c, 5d, 6c, 7a, 8c) were

captured with iQ2.9.1 (Andor) or Metamorph 7.8 (Molecular Devices). The FRAP images (Fig. 3c) and four-color samples of the fixed cells

(Extended Data Fig. 5b, 6a, 6b, 8b) were captured with LAS X 2.0.1 (Leica). The time-lapse images (Fig. 1d) were captured with CV1000

Software 1.06.06 (YOKOGAWA). Cryo-EM images (Fig. 1b; Extended Data Fig. 2d) were collected using SerialEM (V3.7). TEM images (Fig.

2a; Extended Data Fig. 4c, 4d) were collected using AnalySIS 5.0 (Olympus SIS). Droplet formation (Fig. 4d, 4e, 4f; Extended Data Fig. 9c,

9d, 9e) were captured with FV31S-SW 2.3.1.163 (Olympus). Fluorescence images from CLEM (Extended Data Fig. 4b) were collected using

iQ2.9.1 (Andor). MS data were acquired by Xcalibur software 2.2 (Thermo Fisher Scientific).

Data analysis All image analysis and quantification were performed with Metamorph 7.8 (Molecular Devices). All statistical analysis were performed

with GraphPad Prism7 (GraphPad Software). Cryo-EM images were processed using MATLAB R2015b (Mathworks) and TOM software

toolbox (V6.1). K2 frames were aligned using in-house software (K2Align). Tomograms were reconstructed from aligned tilt series by

using the IMOD (4.9.0) software package. PyTom (0.97) was used to identify proteasomes in the tomograms. The resulting

subtomograms were cropped out, CTF- corrected and classified using RELION (3.0). Fluorescence images and TEM images for CLEM were

merged using Icy 1.9.7.0 (Institut Pasteur). MS data were analyzed using Mascot search program (Matrix Science) in Proteome

Discoverer 1.3 (Thermo Fisher Scientific).

For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers.

We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.