Figure 2. Peroxisomal localization of endogenous Miro1 variants. (A) To verify two types antibodies raised to Miro1 and migration of Miro variants in SDS-PAGE, lysates of CHO-K1 cells transfected with nontagged Miro2 (lane 2), Miro1 variants (lanes 3–6), and a mock plasmid (lane 1) were analyzed by immunoblotting. Note that anti–Miro1-ins1 antibody specifically recognizes Miro1-var2 and Miro1-var4 (lanes 5 and 6), whereas anti-Miro1/2 antibody detects all of the Miro1 variants and Miro2 (lanes 2–6). Fraction 2 (lane 7) and half and one-eighth aliquots of fractions 10 and 11, respectively (lanes 8 and 9), from subcellular fractions of HEK cells in B were similarly analyzed. (B) Subcellular fractionation of HEK cells. PNS fraction of HEK cells was fractionated in iodixanol density gradient as described in the Subcellular fractionation section of Materials and methods. Equal-volume aliquots of the collected fractions were analyzed by SDS-PAGE and immunoblotting with antibodies indicated on the left. Cytochrome P450 reductase (P450R), Tom20, and Pex3p are markers for ER, mitochondria (Mito), and peroxisomes (Peroxi), respectively. Arrowheads indicate fractions of highly purified peroxisomes. White lines indicate intervening lanes have been spliced out. (C) Peroxisomal Miro1-var2 is an integral membrane protein. PNS fraction of HEK cells was centrifuged at 3,000 g, and the supernatant (S; 3Kg sup) fraction was divided into three aliquots. One aliquot was directly lysed in Laemmli sample buffer (input). The other two were separately treated with 1 M NaCl and 0.1 M Na2CO3, respectively, and separated into supernatant and pellet (P) fractions. Equal aliquots of respective 3Kg sup fractions and a PNS fraction were analyzed by SDS-PAGE and immunoblotting with antibodies specific for the insertion 1 of Miro1, Tom20, the PMPs Pex14p and Pex13p, and acyl-CoA oxidase (AOx), a peroxisomal matrix enzyme. Only the B chain of AOx that is produced by intraperoxisomal processing of full-length AOx is shown. (D) TMD of Miro1-var2 and Miro1-var4 is required for their localization to peroxisomes. HeLa cells transiently transfected with HA2–Miro1-var2ΔTMD (a and b) and HA2–Miro1-var4ΔTMD (c and d) were immunostained with antibodies to HA and Pex14p as in Fig. 1 C. Bars, 10 µm. (E) Membrane topology of endogenous Miro1-var2 in peroxisomes. The 3Kg sup fraction of HEK cells was incubated for 30 min on ice in the absence (lane 1) or presence of 100 µg/ml trypsin alone (lane 2) and both trypsin and 0.1% Triton X-100 (lane 3). Equal aliquots were analyzed by SDS-PAGE and immunoblotting with indicated antibodies, including the anti-Pex14p antibody specific for the cytosolically faced C-terminal part of Pex14p. (F) Schematic diagram of the topology of Miro1-var2 and Miro1-var4 in peroxisomes. Dark gray boxes, insertions 1 and 2; black and light gray ellipses, a TMD and two GTPase domains, respectively.
Figure 2.

Peroxisomal localization of endogenous Miro1 variants. (A) To verify two types antibodies raised to Miro1 and migration of Miro variants in SDS-PAGE, lysates of CHO-K1 cells transfected with nontagged Miro2 (lane 2), Miro1 variants (lanes 3–6), and a mock plasmid (lane 1) were analyzed by immunoblotting. Note that anti–Miro1-ins1 antibody specifically recognizes Miro1-var2 and Miro1-var4 (lanes 5 and 6), whereas anti-Miro1/2 antibody detects all of the Miro1 variants and Miro2 (lanes 2–6). Fraction 2 (lane 7) and half and one-eighth aliquots of fractions 10 and 11, respectively (lanes 8 and 9), from subcellular fractions of HEK cells in B were similarly analyzed. (B) Subcellular fractionation of HEK cells. PNS fraction of HEK cells was fractionated in iodixanol density gradient as described in the Subcellular fractionation section of Materials and methods. Equal-volume aliquots of the collected fractions were analyzed by SDS-PAGE and immunoblotting with antibodies indicated on the left. Cytochrome P450 reductase (P450R), Tom20, and Pex3p are markers for ER, mitochondria (Mito), and peroxisomes (Peroxi), respectively. Arrowheads indicate fractions of highly purified peroxisomes. White lines indicate intervening lanes have been spliced out. (C) Peroxisomal Miro1-var2 is an integral membrane protein. PNS fraction of HEK cells was centrifuged at 3,000 g, and the supernatant (S; 3Kg sup) fraction was divided into three aliquots. One aliquot was directly lysed in Laemmli sample buffer (input). The other two were separately treated with 1 M NaCl and 0.1 M Na2CO3, respectively, and separated into supernatant and pellet (P) fractions. Equal aliquots of respective 3Kg sup fractions and a PNS fraction were analyzed by SDS-PAGE and immunoblotting with antibodies specific for the insertion 1 of Miro1, Tom20, the PMPs Pex14p and Pex13p, and acyl-CoA oxidase (AOx), a peroxisomal matrix enzyme. Only the B chain of AOx that is produced by intraperoxisomal processing of full-length AOx is shown. (D) TMD of Miro1-var2 and Miro1-var4 is required for their localization to peroxisomes. HeLa cells transiently transfected with HA2–Miro1-var2ΔTMD (a and b) and HA2–Miro1-var4ΔTMD (c and d) were immunostained with antibodies to HA and Pex14p as in Fig. 1 C. Bars, 10 µm. (E) Membrane topology of endogenous Miro1-var2 in peroxisomes. The 3Kg sup fraction of HEK cells was incubated for 30 min on ice in the absence (lane 1) or presence of 100 µg/ml trypsin alone (lane 2) and both trypsin and 0.1% Triton X-100 (lane 3). Equal aliquots were analyzed by SDS-PAGE and immunoblotting with indicated antibodies, including the anti-Pex14p antibody specific for the cytosolically faced C-terminal part of Pex14p. (F) Schematic diagram of the topology of Miro1-var2 and Miro1-var4 in peroxisomes. Dark gray boxes, insertions 1 and 2; black and light gray ellipses, a TMD and two GTPase domains, respectively.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal