List of YFP-tagged transmembrane proteins evaluated for colocalization with mCherry-D4H positive structures. (A)S. pombe cells coexpressing mCherry-D4H, which were treated with 500 µM CK-666 or DMSO as control for 1 h at 25°C. Localization of D4H and YFP-tagged TM proteins was evaluated by spinning disk microscopy. In all cases, D4H localized to the PM in DMSO-treated cells and to internal dots in CK-666–treated ones. TM proteins could be broadly assigned to two classes: those exclusively at the PM in both conditions, and those decorating both PM and internal structures (endomembranes) in both conditions. Upon strong overexpression, several PM-localized TM proteins also decorated internal structures. In the second class, internal signals showed variable extents of colocalization with D4H dots upon CK-666 treatment. Notably, however, none of the TM proteins was exclusively at the PM in DMSO-treated cells and in internal structures upon CK-666 treatment. We also conducted further in-depth analysis by time-lapse imaging of a couple of TM proteins of the second class (Ght3, Liz1), with the aim to capture cointernalizing signals from the PM. However, although colocalization was observed on dynamic internal structures, we did not capture a single convincing event of internalization from the PM. We conclude that the internal signal of these (and likely other) TM proteins was present before CK-666 treatment, and may be partly due to the natural transit of these proteins through the secretory pathway. (B) Images of two exemplary TM proteins YFP-Pma1 localizing exclusively to PM and YFP-Liz1 localizing to PM and internal structures. Scale bar, 2 µm. (C) FM4-64 does not internalize in cells treated with CK-666. (D) Biosensors for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) or phosphatidylserine (PS) are not internalized upon CK-666 treatment. Cells expressing mCherry-D4H and either a PI(4,5)P2 biosensor (2xPH(PLCδ)) or a PS biosensor (LactC2) were treated with 500 µM CK-666 for 1 h. These probes did not colocalize with D4H-positive internal structures. Scale bar, 2 µm.
Figure S3.

List of YFP-tagged transmembrane proteins evaluated for colocalization with mCherry-D4H positive structures. (A) S. pombe cells coexpressing mCherry-D4H, which were treated with 500 µM CK-666 or DMSO as control for 1 h at 25°C. Localization of D4H and YFP-tagged TM proteins was evaluated by spinning disk microscopy. In all cases, D4H localized to the PM in DMSO-treated cells and to internal dots in CK-666–treated ones. TM proteins could be broadly assigned to two classes: those exclusively at the PM in both conditions, and those decorating both PM and internal structures (endomembranes) in both conditions. Upon strong overexpression, several PM-localized TM proteins also decorated internal structures. In the second class, internal signals showed variable extents of colocalization with D4H dots upon CK-666 treatment. Notably, however, none of the TM proteins was exclusively at the PM in DMSO-treated cells and in internal structures upon CK-666 treatment. We also conducted further in-depth analysis by time-lapse imaging of a couple of TM proteins of the second class (Ght3, Liz1), with the aim to capture cointernalizing signals from the PM. However, although colocalization was observed on dynamic internal structures, we did not capture a single convincing event of internalization from the PM. We conclude that the internal signal of these (and likely other) TM proteins was present before CK-666 treatment, and may be partly due to the natural transit of these proteins through the secretory pathway. (B) Images of two exemplary TM proteins YFP-Pma1 localizing exclusively to PM and YFP-Liz1 localizing to PM and internal structures. Scale bar, 2 µm. (C) FM4-64 does not internalize in cells treated with CK-666. (D) Biosensors for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) or phosphatidylserine (PS) are not internalized upon CK-666 treatment. Cells expressing mCherry-D4H and either a PI(4,5)P2 biosensor (2xPH(PLCδ)) or a PS biosensor (LactC2) were treated with 500 µM CK-666 for 1 h. These probes did not colocalize with D4H-positive internal structures. Scale bar, 2 µm.

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