Table 1.
Primers used to generate TMEM68 constructs.
Fig 1.
Sequence and protein domains and phylogenetic relationship of murine TMEM68.
(A) The coding sequence and deduced amino acid sequence of the TMEM68 gene. The nucleotide and predicted amino acid sequences were countered on the left side. Asterisks represent a stop codon. The putative motif I (residues 126–137) and IV (residues 225–234) are shown in the box containing the conserved active sites residues H129 and D135 as well as P229 respectively, which are typed in red color. Residues in the putative motifs II and III (residues 162–171, and residues 194–202) predicted to be important for substrate binding are typed in bold black and red color. (B) The protein domain structure of murine TMEM68. TMEM68 contains a putative acyltransferase domain (residues 111–233) and two transmembrane (TM) domains (residues 51–73, and residues 123–145) shown as green rectangle and black ellipse, respectively. (C) Phylogenetic alignment of murine TMEM68 and other acyltransferases. Protein sequences of glycerophospholipid and diacylglycerol acyltransferase family members (pfam01553 and pfam03982) from human (h), mouse (m) and fruit fly (dm) were aligned using Clustal Ω and clustered using the unweighted pair group method with arithmetic mean.
Fig 2.
Membrane association of TMEM68.
(A) Cellular post-nuclear supernatants (PNS) obtained from COS-7 cells expressing His6-TMEM68 were fractionated by centrifugation (100,000 g) into membrane (mem, pellet) and cytosolic (cyto, supernatant) fractions. Protein detection was performed by immunoblotting using antibodies against the N-terminal His6-tag and GAPDH. (B) Membrane fractions were prepared from COS-7 cells expressing His6-TMEM68 and treated with PBS, 1% SDS, 1%Triton X-100, or 0.1 M sodium carbonate (pH 11.5). After incubation, samples were separated into pellet (P) and supernatant (S) fractions and the presence of His6-TMEM68 and PDI were detected by immunoblotting using anti-His6 and anti-PDI antibodies.
Fig 3.
Membrane orientation of the N- and C-terminus of TMEM68.
Total membranes obtained from the COS-7 cells expressing His6-TMEM68 (A) and TMEM68-FLAG (B) were incubated in the absence or presence of proteinase K (PK) and/or 1% Triton X-100 (TX) and subjected to immunoblotting using antibodies against His6, FLAG, and PDI.
Fig 4.
Subcellular localization of TMEM68-GFP.
(A) Detection of TMEM68-GFP and GFP expression by immunoblotting. 48-h post transfection, cells were harvested, homogenized, and subjected to immunoblotting using an anti-GFP antibody. (B) Subcellular localization of TMEM68-GFP in mammalian cells. COS-7 cells were transfected with plasmids encoding for GFP, TMEM68-GFP, and DsRed-ER as indicated and imaged by confocal fluorescence microscopy. For the detection of LDs, cells were incubated with OA and stained with LipidTOX Deep Red. Scale bar = 10 μm. Figures are representative of three separate experiments.
Fig 5.
Contribution of TMDs to ER targeting of TMEM68.
(A) Scheme of GFP-tagged TMEM68 mutant proteins harboring deletions of the first (ΔTMD1), the second (ΔTMD2) or both (ΔTMD1+2) TMDs (shown as black boxes) (B) COS-7 cells were co-transfected with constructs encoding for ΔTMD1-GFP, ΔTMD2-GFP or ΔTMD1+2-GFP and the ER marker, DsRed-ER. 48-h post transfection, cells were analyzed by a confocal laser scanning microscopy. Scale bar = 10 μm. Figures are representative of three separate experiments. (C) Post-nuclear supernatants of COS-7 cells expressing TMEM68 mutant proteins were fractionated by centrifugation into membrane (mem, pellet) and cytosolic (cyto, supernatant) fractions and analyzed by immunoblotting using antibodies against GFP or the ER marker calnexin.
Fig 6.
Targeting of GFP to the ER by the first TMD of TMEM68.
(A) Scheme of the constructs expressing the first (TMD1), the second (TMD2), and both TMDs (TMD1+2) tagged with GFP. Black boxes represent the first and second TMDs of TMEM68. (B) Post-nuclear supernatants of COS-7 cells expressing TMD1-GFP, TMD2-GFP or TMD1+2-GFP were fractionated into cytosolic and membrane fractions and analyzed by immunoblotting using antibodies against GFP or the ER protein calnexin. (C) COS-7 cells were co-transfected with TMD1-GFP, TMD2-GFP or TMD1+2-GFP and the ER marker DsRed–ER as indicated and visualized by a confocal fluorescence microscopy. Scale bar = 10 μm. Figures are representative of three separate experiments.
Fig 7.
Expression of TMEM68 in adult murine tissues.
Relative transcript levels of TMEM68 were measured by quantitative RT-qPCR using 36B4 as a reference gene. The y-axis represents relative mRNA expression levels of TMEM68. White adipose tissue (WAT), Brown adipose tissue (BAT), skeletal muscle (SM), cardiac muscle (CM). *, p<0.05; n = 3.