Supplementary Figures
Figure S1. The interaction between ULK1 and ATG13 is severely compromised by deletion of
the TLQ480 motif. Atg13 KO MEFs retrovirally transfected with empty vector or cDNA
encoding HA-ATG13 or HA-ATG133AA∆ were labeled with light (R0K0), medium (R6K4) or heavy
(R10K8) arginine/lysine. Cells were lysed and HA immunopurifications were performed.
Purified proteins were analyzed by mass spectrometry as described in the supplementary
Material and Methods section. Unnormalized SILAC ratios for ULK1 obtained from MaxQuant
were normalized to the ratio of ATG13.
Figure S2. ULK1 cannot be copurified with RB1CC1 or ATG101 in the absence of full-length
ATG13. (A) Wild-type or Atg13 KO MEFs were lysed and anti-RB1CC1 immunopurifications
were performed. Cleared cellular lysates (CCL) and purified proteins were subjected to SDSPAGE and analyzed by immunoblotting for RB1CC1, ULK1, or ATG13. (B) Atg13 KO MEFs
retrovirally transfected with empty vector or cDNA encoding HA-ATG13 (WT) or HA-ATG133AA∆
(3AA∆) were incubated in full medium or starvation medium (EBSS). Cells were lysed and antiULK1 (middle panels) or anti-ATG101 (right panels) immunopurifications were performed.
CCLs and purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for
ULK1, ATG13, or ATG101.
Figure S3. Amino acid alignment of ATG13/Atg13 proteins. (A) Full length ATG13/Atg13
proteins were aligned using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/).
Alignments were coloured using Jalview software. Amino acid sequences derive from the
following species: Caenorhabditis elegans (UniProt ID P34379), Drosophila melanogaster
(Q9VHR6), Danio rerio (Q7SYE0), Gallus gallus (E1BQS0), Mus musculus (Q91YI1), Bos taurus
(Q08DY8), Homo sapiens (O75143), Felis catus (M3WJ75), Canis familiaris (F1P6L1),
Saccharomyces cerevisiae (Q06628), and Schizosaccharomyces pombe (O36019). (B) Cterminal parts of the indicated ATG13 proteins were aligned as in (A).
Figure S4. Partial restoration of autophagic flux by ATG133AA∆ depends on the BECN1PIK3C3/VPS34 complex. (A and B) Atg13 KO MEFs retrovirally transfected with empty vector
or cDNA encoding HA-ATG13 or HA-ATG133AA∆ were transiently transfected with the indicated
siRNAs. Cells were incubated in full medium or starvation medium (EBSS) for 2 h in the absence
or presence of 40 nM bafilomycin A1 (Baf A1). Cells were lysed and whole cellular lysates were
subjected to SDS-PAGE and analyzed by immunoblotting for ULK1, SQSTM1, GAPDH, BECN1
or LC3. (C) MEFs described in (A and B) were incubated in full medium or starvation medium
(EBSS) for 2 h in the absence or presence of 40 nM bafilomycin A1 (Baf A1) and/or 10 mM 3MA. Cells were lysed and whole cellular lysates were subjected to SDS-PAGE and analyzed by
immunoblotting for LC3 or ACTB.
Supplementary Materials and Methods
siRNA-mediated knockdown of Ulk1, Ulk2 and Becn1
MEFs were seeded on a 24-well plate overnight. Cells were transfected using Lipofectamine
RNAiMAX (Life Technologies, 13778-150) according to the manufacturer’s instructions with
20 nM ON-TARGETplus SMARTpool® siRNA (Dharmacon, Thermo Scientific) targeting Ulk1
(L-040155-00-0005), Ulk2 (L-040619-00-0005) or Becn1 (L-055895-00-0005), or a nontargeting control pool (D-001810-10-05). Knockdown efficiency was analyzed by
immunoblotting.
Mass Spectrometry
Sample preparation
Protein mixtures were reduced with Dithiothreitol (Sigma Aldrich, 43815) and alkylated with
Iodacetamide (Sigma Aldrich, I11A49) and subsequently separated by SDS-PAGE using 4-12%
Bis-Tris mini gradient gels (NuPAGE, Life Technologies, NP0335BOX). The gel lanes were cut
into 10 equal slices, proteins were in-gel digested with trypsin (Promega, V5113),1 and the
resulting peptide mixtures were processed on StageTips using C18 material (3M Empore C18,
IVA Analysetechnik, 2215) as described previously.2
MS analysis
Mass spectrometric measurements were performed essentially as described 3 on an LTQ
Orbitrap XL mass spectrometer (Serial #: 01599B, Thermo Fisher Scientific, Langenselbold,
Germany) coupled to an Agilent 1200 nanoflow-HPLC (Agilent Technologies, Waldbronn,
Germany) or a NanoLC Ultra (Serial #: 11-01-15-215, Eksigent, Darmstadt, Germany). HPLCcolumn tips (fused silica) with 75 µm inner diameter (New Objective, FS360-75-10-N-5-C25)
were self-packed with Reprosil-Pur 120 ODS-3 (Dr. Maisch, r13.aq) to a length of 20 cm.
Samples were applied directly onto the column without pre-column. A gradient of A (0.5%
acetic acid [high purity, LGC Promochem, 33209-1L-GL] in water) and B (0.5% acetic acid in
80% acetonitrile [LC-MS grade, Promochem, SO-9340-B025] in water) with increasing organic
proportion was used for peptide separation (loading of sample with 2% B; separation ramp:
from 10-30% B within 80 min). The flow rate was 250 nl/min and for sample application 500
nl/min. The mass spectrometer was operated in the data-dependent mode and switched
automatically between MS (max. of 1x106 ions) and MS/MS. Each MS scan was followed by a
maximum of 5 MS/MS scans in the linear ion trap using normalized collision energy of 35%
and a target value of 5000. Parent ions with a charge state from z=1 and unassigned charge
states were excluded for fragmentation. The mass range for MS was m/z = 370 to 2000. The
resolution was set to 60,000. Mass-spectrometric parameters were as follows: spray voltage
2.3 kV; no sheath and auxiliary gas flow; ion-transfer tube temperature 125°C.
Identification of proteins and protein ratio assignment using MaxQuant
The MS raw data files were uploaded into the MaxQuant software4 version 1.3.0.5., which
performs peak detection and generates peak lists of mass error-corrected peptides using the
following parameters: carbamidomethylcysteine was set as fixed modification, methionine
oxidation and protein amino-terminal acetylation were set as variable modifications. Three
miss-cleavages were allowed, enzyme specificity was trypsin/P + DP, and the MS/MS tolerance
was set to 0.5 Da. Peak lists were searched by Andromeda for peptide identification using a
Uniprot mouse database from September 2012 containing common contaminants such as
keratins and enzymes used for in-gel digestion (55299 entries). The database was
complemented by the sequence of human ATG13 (O75143). Peptide lists were further used
by MaxQuant to identify and relatively quantify proteins using the following parameters:
peptide, and protein false discovery rates were set to 0.01, maximum peptide posterior error
probability (PEP) was set to 1, minimum peptide length was set to 7, the PEP was based on
Andromeda score, minimum number of peptides for protein identification was set to one,
which must be unique, and for protein quantitation a ratio count of 2 was required. Identified
proteins were requantified.
Data analysis
Unnormalized SILAC ratios for ULK1 obtained from MaxQuant were normalized to the ratio of
ATG13.
Supplementary References
1.
2.
3.
4.
Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass
spectrometric characterization of proteins and proteomes. Nat Protoc 2006; 1:2856-60.
Rappsilber J, Mann M, Ishihama Y. Protocol for micro-purification, enrichment, prefractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2007;
2:1896-906.
Sprenger A, Weber S, Zarai M, Engelke R, Nascimento JM, Gretzmeier C, Hilpert M,
Boerries M, Has C, Busch H, et al. Consistency of the proteome in primary human
keratinocytes with respect to gender, age, and skin localization. Mol Cell Proteomics
2013; 12:2509-21.
Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized
p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol
2008; 26:1367-72.