New Phytologist Supporting Information Figs S1–S7
S-acylation anchors Remorin proteins to the plasma membrane but does not
primarily determine their localization in membrane micro-domains
Sebastian S. A. Konrad, Claudia Popp, Thomas F. Stratil, Iris K. Jarsch, Veronika
Thallmair, Jessica Folgmann, Macarena Marín and Thomas Ott
The following Supporting Information is available for this article:
1
Supporting Information Fig. S1 Protein interaction scores within the SYMREM1
protein and free YFP in root epidermal cells. (a) Ab initio modelling of the
SYMREM1 protein and colour-coded representation of putative regions that may
contribute to protein interactions. Models for the N- and C-terminal regions were
constructed independently and fused subsequently. Details can be found in the
Materials and Methods section. ID, intrinsically disordered (b) M. truncatula root
epidermal cell expressing a free YFP protein. The image shows a maximum intensity
projection of a z-stack. n, nucleus; cyt, cytoplasmic strands. Bar, 5 μm.
2
Supporting Information Fig. S2 Analysis of RemCA-mediated PM-binding throughout the Remorin protein
family. (a–d) Four out of 16 RemCA peptides were sufficient to target the fluorophore almost exclusively to the
plasma membrane of N. benthamiana root epidermal cells. (e–p) Representative images of the remaining twelve
RemCA peptides show at least partial cytoplasmatic and nuclear labelling. Bars, 20 μm. (q) Western Blot on total
protein extracts from N. benthamiana leaves expressing different RemCA peptides. Double bands indicate partial
cleavage of the fusion protein. Samples were compared to free YFP. (r) Microsomal fractionations of total protein
extracts were performed to assess partial cleavage of the respective constructs. sol., soluble protein fraction; μ,
microsomal fraction.
3
Supporting Information Fig. S3 Western Blots and microsomal fractionations of
wild-type and mutated SYMREM1 fusion proteins. (a) Microsomal fractions were
obtained from N. benthamiana leaves expressing YFP-SYMREM1 and YFPSYMREM1C197A fusion proteins. Both wild-type and the mutant variant were found in
the microsomal fraction, indicating that they remained at the plasma membrane. (b)
Microsomal fractions of wild-type and mutated SYMREM1 showed a band shift
pattern. Western blots were probed with α-GFP antibodies.
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Supporting
Information Fig. S4
Co-localization
studies for different
mutant variants.
YFP-tagged proteins
were expressed in N.
benthamiana leaf
epidermal cells and
counterstained with
FM4-64. All images
show maximum
projections of zstacks taken of secant
planes. Bars, 5 μm.
5
Supporting Information Fig. S5 Biotin switch assay and quantification. (a) Control
experiment to prove functionality of the assay. Full-length At3g61260 is S-acylated as
previously demonstrated in Hemsely et al. (2013). S-acylation of proteins is indicated
by the presence of a band in the elution fraction of the hydroxylamine treated samples
(+). (b) Quantification of the Western Blot in (a) using ImageJ. (c, d) Quantification
of Western Blots shown in Fig. 5. Values were normalized to background levels.
double, double mutant.
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Supporting Information Fig. S6 Western blot analysis of YFP-SYMREM1
constructs expressed in yeast. Free YFP was expressed as a control for the
fractionation procedure. Microsomal fractions were obtained and the corresponding
SYMREM1 proteins were detected using a α-GFP antibody. μ, microsomal fraction;
sol., soluble proteins.
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Supporting Information Fig. S7 Proposed model for membrane-binding of Remorin
proteins. Remorins are soluble proteins (a) that are initially immobilized at the PM via
interactions with a membrane resident protein (b) or by direct protein-lipid
interactions via their C-terminal hydrophobic core (e). (c) Interaction with a protein
partner leads to partial disorder-to-order transition of the intrinsically disordered Nterminal region. This may involve protein phosphorylation (red). (d, e) A membranelocalized protein acyl transferase (PAT) S-acylates (green line) C-terminal cysteine
residues of Remorins and possibly others throughout the protein. This lipidation
tightly binds the protein to the PM and may confer some degree of specificity to
sterol-rich sites (blue) in the PM. Oligomerization of Remorins contributes to the
formation of larger domain platforms (hypothetical).
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