Fig. 2: A short peptide motif enhances self-assembly to drive focal enrichment after import.

a Scheme of the transcriptional pulse-chase experiment. Cells were analyzed at indicated time points by super-resolution microscopy using SIM. GFP-Mac3 was compared to GFP-Aox1 (cyan) in strains containing mCherry-SKL. Scale bar: 0.5 µm. b Quantification of colocalization data derived from epifluorescence microscopy depicted in Fig. S3b based on three independent biological replicates. Samples were analyzed directly after (t0), 3 h after (t3), and 6 h after glucose addition (t6). Quantifications show Pearson’s correlation coefficients of GFP and mCherry signals for indicated strains. Center line, median. Significance assessed by an unpaired, two-sided Student’s t-test. **** refer to a p value lower than or equal to 0.0001. c Western blot analysis following the stability of GFP-Mac3 and GFP-Aox1 in a time course experiment. d C-terminal 12 amino acids containing the PTS1 motifs. Cells expressing GFP tagged variants were analyzed by SIM. Representative images of cells are shown. Full images are shown as overlays of two channels. For insets single channels and merged channels are shown. Scale bars: 5 µm. e GFP fused to Thr-Ile-Ile-Val (TIIV) and the PTS1 Ser-Lys-Leu (SKL) was co-expressed with mCherry-SKL. Scale bars: 5 µm. f Cells expressing PTS2-GFP reporter proteins either with or without a C-terminal TIIV motif (cyan) were co-expressed with mCherry-SKL. Quantification showing Pearson’s correlation coefficients of GFP and mCherry signals in Fig. S5e. Scale bars: 5 µm. g Mac3-V₅₇₀R was tagged with GFP (cyan) and localization was analyzed by epifluorescence microscopy. Quantifications in Fig. S6d. Scale bars: 5 µm. h The TIIV motif of Mac3 is embedded in an aggregation prone region. Aggregation of 6xHis-tagged (i) versions of Mac3 and (j) versions of GFP-TIIV and GFP-TIIR was measured over time. Plotted are the means of three replicates for each protein. Different colors denote single experiments. Center line, mean; error bars; standard error of the mean. Significance assessed with an unpaired, two-sided Student’s t-test. *** refer to a p value of 0.0003 (i) and (j). Source data underlying b, c, i, j are provided as a Source data file. GFP, cyan; mCherry, magenta.