Supplementary Figure 1. Phylogeny and TM prediction of MLC1. ;Supplementary Figure 2. The amino acid sequence of hMLC1-GFP. ;Supplementary Figure 3. Predicted structure of MLC1 proteins;Supplementary Figure 4. Purification and oligomerization of ckMLC1. ;Supplementary Figure 5. Determination of the molecular weight of the control proteins. from Transmembrane topology and oligomeric nature of an astrocytic membrane protein, MLC1
Creators: name: Hwang, Junmo
name: Park, Kunwoong
name: Lee, Ga-Young
name: Yoon, Bo Young
name: Kim, Hyunmin
name: Roh, Sung Hoon
name: Lee, Byoung-Cheol
name: Kim, Kipom
name: Lim, Hyun-Ho
Publisher: name: The Royal Society
Year: 2021
Subjects: Biophysics
Biochemistry
Cell Biology
Neuroscience

description: (A) Phylogenetic analysis and amino acid sequence conservation of MLC1 orthologues. The phylogenetic tree was generated by the maximum-likelihood phylogeny analysis, PhyML in SEAVIEW software [62]. Protein accession numbers for indicated MLC1 orthologues are human (NP_631941.1), chimpanzee (XP_515212.3), rhesus monkey (XP_001111622.1), dog (XP_848790.2), cow (NP_001070420.1), mouse (NP_573504.1), rat (NP_001101575.1), chicken (XP_004937559.1), zebrafish (NP_001098409.1), and clawed frog (NP_001072921.1). (B) TM domains of hMLC1 are predicted by different algorithms (TOPCONS web server, https://topcons.cbr.su.se) [25].;The c-Myc tag (N-EQKLISEEDL-C) insertion sites in the putative loops are indicated as blue arrowheads and marked as L1 to L7. Single amino acid substitutions found in patients are indicated by brown (in the cytosolic domain), red (in the TM domain), and magenta (in the loop region) letters. Green letters indicate the enhanced green fluorescence protein. The TM domains are shaded yellow and marked with underlines.;(A) AlphaFold structure prediction of human MLC1 protein. A per-residue confidence score (pLDDT, 0 to 100) is supplemented to indicate model confidence. Note that the model confidences of helices are relatively high (cyan and blue), but that of the N-terminal part is very low (orange). (B) Merged structures of MLC1 proteins from human (blue), mouse (cyan), and zebrafish (grey). The low-confidence N-terminal parts were removed for clarification. The alpha helices are labeled as h1 to h8, and the C-terminal short helix is named as h-ct. The merged structures were generated by using PyMOL software ver 2.4.0 (The PyMOL Molecular Graphics System, Version 2.0, Schrodinger, LLC).;Glutaraldehyde cross-linking experiments with ckMLC1 showed an oligomerized pattern of ckMLC1 in detergent micelles (A) and liposomes (B). Lane 1: without glutaraldehyde. Lane 2: crosslinking with glutaraldehyde. Lane 3: crosslinking with glutaraldehyde in the presence of 2% (w/v) SDS. After the crosslinking reaction for 1 min at 23°C, the samples were subjected to SDS-PAGE, and protein bands were visualized by Coomassie blue staining (A) or western blotting (B, with anti-penta-His antibody). Empty circles mark the oligomeric compositions of indicated protein bands. (C) FPLC profile of purified C-terminal 6X His-tagged ckMLC1 and CLC-ec1.;Bovine serum albumin (BSA) (A), CLC-ec1_P150V (B), and CLC-ck2 (C) were subjected to SEC-MALS-RI. The estimated sizes are indicated.