The order of enzymatic activity across Golgi cisternae is essential for complex molecule biosynthesis. However, an inability to separate Golgi cisternae has meant the cisternal distribution of most resident proteins, and their underlying localization mechanisms, are unknown. Here, we exploit differences in surface charge of intact cisternae to perform separation of early to late Golgi sub-compartments. We determine protein and glycan abundance profiles across the Golgi; over 390 resident proteins are identified, including 136 new additions, with over 180 cisternal assignments. These assignments provide a means to better understand the functional roles of Golgi proteins and how they operate sequentially. Protein and glycan distributions are validated in vivo, using high resolution microscopy. Results reveal distinct functional compartmentalization among resident Golgi proteins. Analysis of transmembrane proteins shows several sequence-based characteristics relating to pI, hydrophobicity, Ser abundance and Phe bilayer asymmetry that change across the Golgi. Overall our results suggest that a continuum of TM features, rather than discrete rules, guide proteins to earlier or later locations within the Golgi stack.
Supplemental Dataset 1
Supplemental Dataset 1: Resident organelle proteomes from LOPIT experiments (Fig. 2, S1) after SVM-based classification. Proteins are identified by their AGI accession code for the TAIR database. Proteins annotated with an 'x' formed the training datasets used for SVM classification and those annotated as '-' were newly classified as organelle residents. Classification thresholds and SVM parameters were as described in Experimental Procedures.
Table S1.xlsx
Supplemental Dataset 2
Supplemental Dataset 2. Additional information for monoclonal antibodies, polysaccharide epitopes and protein targets featured in Fig. 3. Antibody names in column A refer to antibodies featured in Fig. 3a, b, and c. For each antibody the original publication reference (column B), epitope strucutre (column C) and polysaccaride containing that epitope (column D) is given. Group members (Fig. 3c) are detailed in column F. For peptide markers assayed in Fig. 3e, localization and relevant localization references are supplied in columns H-J.
Table S2.xlsx
Supplemental Dataset 3
Supplemental Dataset 3. Protein lists for sub-Golgi proteomes. Proteins (column A) are annotated with sub-Golgi classification (column B) , MapMan functional classifications (column D) and gene descriptions (column E). 'x' (column C) denotes which proteins formed the SVM training set shown in Fig. 4c that was used in the final classification in Fig. 4d. Proteins were annotated with functional categorization and gene descriptions using the MapMan tool (http://mapman.gabipd.org/mapman). Peptide spectral matches, averaged from replicates, give an overview of protein abundance columns G - J). Further quantitative information is available at the ProteomeXchange Consortium via the PRIDE partner repository (PXD004596, PXD009978).
Table S3.xlsx
Supplemental_Dataset_4_part1of3
Image regions analyzed in Figure 5C
Supplemental_Dataset_S4_part1.zip
Supplemental_Dataset_S4_part2of3
Image regions analyzed in Figure 5C
Supplemental_Dataset_S4_part2.zip
Supplemental_Dataset_S4_part3of3
Image regions analyzed in Figure 5C
Supplemental_Dataset_S4_part3.zip
