Extended Data Fig. 2. ARL6IP1 and FAM134B share structural features and interact.
a) Predicted alignment error of the relative organization of key structural elements of ARL6IP1 shown in Fig. 2b. b) Helical wheel representation of two cytosolic helical segments with characteristics of amphipathic helices with a large hydrophobic moment and net positive charge (blue circle)52 . c) Pairwise sequence alignment of ARL6IP1 and FAM134B indicates preserved RHDs, i.e. two helical hairpins TM1+2 and TM3+4 (grey), and two amphipathic helices, AHL and AHC (yellow). Both proteins harbour several predicted ubiquitination sites (red triangles). d) Doxycycline induced HA-ARL6IP1 interacts with endogenous FAM134B in U2OS cells (1 exp.). e) ARL6IP1 co-precipitates with all known members of the FAM134 family of proteins (1 exp.). The interaction with FAM134B requires the N-terminal part of FAM134B with its first RHD and is independent of its C-terminal coiled-coil domain. Cells were transfected with Myc-ARL6IP1 and the indicated GFP-tagged FAM134B deletion constructs. ATL3, another ER-protein characterised by a RHD, served as a negative control. f) The central part of ARL6IP1 with both helical hairpins is involved in the interaction with FAM134B (1 exp.). HEK293T cells were transfected with the indicated HA-tagged ARL6IP1 deletion constructs. WT and variant proteins were pulled down and the endogenous binding partner FAM134B was detected. The deletion sites are indicated as black bars. The replaced lysines for the ARL6IP1-7KR variant are indicated by red bars. Variants marked with * lack the terminal KKNE signal (white bar). g) The central part of FAM134B with both helical hairpins is required for the interaction with ARL6IP1 (1exp.). HEK293T cells were transfected with the indicated HA-tagged FAM134B deletion constructs. The deletion sites are indicated as black lines. WT and variant proteins were pulled down and the endogenous binding partner ARL6IP1 detected.