In order to determine whether the MRPL12 mutation also induced changes in composition and assembly of the mitochondrial ribosomal large and small subunits, mitochondrial lysates from cultured fibroblasts (subject and control) were fractionated on isokinetic sucrose gradients (10–30%, as in Ref. [47]). If assembly of either the large subunit or the entire ribosome was affected then the distribution of individual ribosomal proteins would change within the gradient profile. On analysis MRPL12 from the patient was substantially decreased in all fractions but detectable in the fractions consistent with mt-LSU; however it was noticeably absent from the free pool (fractions 1 and 2, Fig. 5). This was in contrast to the control that exhibited a pool of free MRPL12, which has been reported to interact with POLRMT [56]. MRPL3 was also slightly reduced in subject cells but remained in fractions consistent with the large subunit. The MRPL12 mutation impacted more modestly on the small ribosomal subunit, with DAP3 apparently unaffected and MRPS18B found in lower amounts only in fractions 4 and 5 but otherwise with similar steady state levels and distribution profile compared to control. Since POLRMT and MRPL12 have been published as interactors, we analyzed both the steady state level and gradient distribution of POLRMT to see if these were affected by the MRPL12 mutation. Overall levels in the subject sample were decreased to 63% of control value (Fig. 5B) but distribution in the gradient appeared largely unaffected with the exception of fraction 11, where levels were lower than control (Fig. 5A bottom panels).
isokinetic sucrose gradients
10–30%
%
control value
levels in the subject sample
63%
%