Figure 2. ARL4D(T35N) localized to the mitochondrial inner membrane.

(A) The fractionation of ARL4D(Q80L) or ARL4D(T35N)-expressing cells by gradient centrifugation. The postnuclear supernatants of COS-7 cells expressing ARL4D(Q80L) or ARL4D(T35N) obtained at 48 h after transfection were fractionated on iodixanol gradients as described in the Materials and Methods. Samples collected from the top of each indicated fraction were analyzed by Western blot with antibodies against ARL4D, Tim23, syntaxin-6 (Stx6), and α-tubulin. The protein levels in each fraction are reported as the percentage of total protein recovered, as determined by densitometry. (B) The mitochondria-enriched membrane fractions of COS cells expressing un-tagged ARL4D(T35N) were incubated with or without (−) 50 µg/ml proteinase K (PK) in the presence or absence of 0.1% Triton X-100 (TX-100). The samples were precipitated with TCA and analyzed by Western blotting with antibodies against ARL4D, Tim23, VDAC, and pyruvate dehydrogenase (PDH)-E2. (C) The mitochondria-enriched membrane fractions from COS cells expressing untagged ARL4D(T35N) were treated with different digitonin/protein ratios, varying from 1 to 20, as described in the Materials and Methods. The proteins present in the membrane fractions after digitonin treatment were analyzed by Western blotting. (D) The mitochondria isolated from COS cells expressing untagged ARL4D(T35N) were treated with 0.1% Triton X-100, 0.1 M Na₂CO₃ (pH 12) or buffer (Mock) and centrifuged to separate the soluble supernatants (S) and membrane pellets (P). The samples were analyzed by Western blotting with antibodies against the indicated proteins. (E) Cryosections of COS-7 cells that had been transfected with the plasmid encoding ARL4D(T35N)-myc were processed for immunogold EM to detect ARL4D(T35N)-myc. Gold particles indicating the presence of ARL4D(T35N) (gold particles, arrows) decorated the mitochondrial inner membrane. Bar, 100 nm.