Figure 3. Tetracyclines mediate MALSU1 recruitment to the mitoribosome large subunit.

• A, B
  (A) Isolated mitochondria and mitoribosomes from Expi293F exhibit enrichment of MALSU1 in doxycycline treated cultures, that is (B) associated with the large subunit of the mitoribosomes evidenced by sucrose gradient sedimentation (15–30% sucrose). Sedimentation of large and small subunit were assessed by MRPL4 and MRPS16, respectively. Duplicates of mitochondria and mitoribosomes in (A, n = 2 independent experiments) are the inputs for (B). Note shift in MALSU1 and MRPL4 upon doxycycline treatment.
• C
  Isolation of crude mitochondria from doxycycline (1 μM) treated ND1 cells (non‐target control (NTC) and sgMALSU1). Note increased MALSU1 expression in doxycycline‐treated mitochondria.
• D
  Crude mitochondria from ND1‐NTC cells were sucrose gradient (15–30%) sedimented and analyzed for MALSU1 as in (B). Note shift and enrichment of MALSU1 and MRPL4 with doxycycline treatment.
• E
  Quantitation of MALSU1 elution profile in sucrose gradients of crude mitochondria from ND1 cells derived from (D), normalized to input signal of MALSU1.
• F
  Sucrose gradient (15–30%) sedimentation of MALSU1‐depleted ND1 cells with or without doxycycline treatment. Crude mitochondria from (C) are inputs for (D) and (F).
• G, H
  Cell fractionation of (G) ND1 and (H) Expi293F suspension cultures treated with doxycycline. Note enrichment of MALSU1 specifically in mitochondrial fractions and decreases in mitochondrial markers TOM70 and Cytochrome C at the whole cell level.
• I
  ND1 cells treated with increasing concentrations of doxycycline illustrate dose‐dependent increases in MALSU1 specifically at the mitochondria without changes in total mitochondria (VDAC, LRPPRC).

Source data are available online for this figure.