Extended Data Fig. 6: Topology and moonlighting analysis.

a–c, Predicted versus annotated proportion of protein exposed to the cytosol or lumen for ER transmembrane proteins. a, Hypothetical examples of proteins with varying proportions of their sequence exposed to the cytosol or lumen. The extent of labelling by cytosolic or lumenal baits should be directly related to proportion of the sequence, and hence lysine residues available for biotinylation, exposed to the respective faces of the membrane that the protein spans. b, All transmembrane domain containing prey proteins localized to the cytosolic face of the ER (NMF compartments 3 and 15) and the lumenal face (NMF compartment 6), were assigned a CLR score on the basis of their NMF profile (313 proteins). The CLR score of a prey is calculated by taking the score in the cytosolic facing compartment/maximum score in that compartment and subtracting the corresponding score in the lumenal compartment. A score closer to 1 would indicate a protein with a signature at the cytosolic face of the ER membrane but little or no signature in the lumen and a score of −1 would indicate the opposite. A similar sequence-based score was calculated as the fraction of the sequence annotated as cytosolic minus the fraction that is lumenal according to UniProt. KTN1 is mis-annotated in UniProt⁵⁶ and should have a sequence score of +0.9742. c, Three example of proteins and their topology, CLR and sequence scores. Green examples have predictions matching annotated topology. d, e, Moonlighting and connections between compartments. d, Primary and secondary localizations of moonlighting preys. Preys with a score of at least 0.15 in each of two non-contiguous NMF compartments were considered to moonlight (a list of non-contiguous compartments is in Supplementary Table 15). The number of preys with a primary localization defined on the vertical axis and a secondary localization defined on the horizontal axis is shown (maximum 18). e, Inter-compartment edges were counted for each NMF rank/compartment. An interaction edge was defined between prey pairs having a correlation score across all NMF compartments of at least 0.9. Edges were then defined as ‘intra-compartment’ (if the primary localization for the two preys was the same compartment) or ‘inter-compartment’ (if the primary localization for the two preys was in different compartments) (Supplementary Table 15). Most organelles displayed a much greater proportion of intra-compartment interactions, with the extreme case of the mitochondrial matrix having only 15 inter-compartmental edges out of a total of 37,387 edges. The proportion of inter-compartment edges from the source to each target compartment is shown here. Inter-compartmental edges generally conformed with expectations, for example with edges from the chromatin compartment connecting to other nuclear substructures with which they may exchange components. The NMF rank number is shown in brackets next to the source compartment name.

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