Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling

Supplemental Data

  • Supplementary Figure 1 (.pdf, 820 KB) - Supplementary Figure 1: Assignment of 15N-labeled partners to unlabelled peptide species identified by MaxQuant. (A) Workflow of linking data from ?evidence.txt? and ?matched features.txt?. (B) Mass spectrum of an unlabelled and 15N-labeled peptide ion with highlighted sampling window to identify the 15N-form within a 1:1 mixture of labeled and unlabeled membrane protein. Sampling window and sampled 15N ion peak is indicated in red. Sampled 14N peak is indicated in blue.
  • Supplementary Figure 2 (.pdf, 1.1 MB) - Supplementary Figure 2: K-means clustering of wild type and mutant protein abundance profiles. (A) overview of the six clusters. (B) Distribution of wild type and mutant proteins across the different clusters. (C) Overlap of the identified candidates based on individual altered distribution profiles or the clustering method (?clustering?).
  • Supplementary Figure 3 (.pdf, 419 KB) - Supplementary Figure 3: Localization of GFP-PIP2A in transiently transformed mesophyll protoplasts. (A) Single sections (top row) and maximum projections (bottom row) of representative protoplasts for each genotype. GFP fluorescence is shown in green, autofluorescence of chlorophyll is shown in red. Scale bars represent 10 ?m. (B) Distribution of the GFP-PIP2A locations to different subcellular compartments in individual transformation events to plasma membrane (PM, as represented by col-0 in panel A) or ?other? cell internal compartments (e.g. as represented by ap-4? in panel A). The numbers of images scored with at least 10 fluorescent protoplasts eachwere 18 for ap-3?, 22 for col-0 and 24 for ap-4?. Mean values indicate the location counts per image and are shown with standard error.
  • Supplementary Figure 4 (.pdf, 1.8 MB) - Supplementary Figure 4: Distribution across fractions for proteins with known mis-targeting in the ap3-? mutant. (A) PIN7, (B) PIN3, (C) BRI1 (D) VSP1. Values describe the mean with standard deviation of four independent gradient preparations for col-0 and two gradients for ap-3? and ap-4? mutants.
  • Supplementary Figure 5 (.pdf, 144.4 MB) - Supplementary Figure 5: Spectra of identified phosphopeptides. Spectra were plotted based on the MaxQuant results.
  • Supplementary Table 1 (.xlsx, 14 KB) - Supplementary Table 1: Reproducibility of the step gradient preparations regarding sucrose concentration at the interphase, protein concentration and the numbers of identified peptide sequences and proteins.
  • Supplementary Table 2 (.xlsx, 20 KB) - Supplementary Table 2: List of marker proteins and their predicted and confirmed sub-cellular localizations used across the gradient to assign the interphases to organelle-enriched fractions.
  • Supplementary Table 3 (.xlsx, 133 KB) - Supplementary Table 3: List of proteins with altered distribution profiles in replicated experiments.
  • Supplementary Table 4 (.xlsx, 38 KB) - Supplementary Table 4: List of phosphopeptides and pairwise comparison between wild type and AP-complex mutants.
  • Supplementary Table 5 (.xlsx, 21.7 MB) - Supplementary Table 5: List of all identified protein groups including numbers of peptides, proteins per protein group and sequence coverage of each protein.
  • Supplementary Materials Description (.pdf, 417 KB) - supplementary materials description