un-degraded forms of the large and small rRNA subunits (Figure 5A), and others (e.g. mitochondrial genome main- tenance and chaperons) (Supplementary Table S1). In the following we will refer to this complex as the ‘RMT Figure 3. A homogenous, stable RMT complex. A total of 50 mg protein was loaded per lane, and after overnight electrophoresis (140 V and 9 mA; 4 C) the gel was either stained with silver nitrate (BioRad silver staining kit) in (A), and Serva Blue G250 in (B). (A) Aliquots of fractions 7, 8 and 9 (with mt-RNase P activity) from a 5.5% preparative NEC were analyzed by slab-gel NEC (4–14%) for complex composition. (B) The RMT complex (fraction 38 from 7.5% NEC) is to some degree ( 50%) not dissociated by treatment (30 min) with detergent [digitonin/protein ratio (2 g/g)]. Nucleic Acids Research, 2012, Vol. 40, No. 4 1733 complex’ (for RNA processing, metabolism, translation complex). A total of 564 physical protein–protein inter- actions are listed among proteins of the RMT complex in the BioGRID and SGD databases, in support of their organization in a complex. Besides Rpm2p and tRNA Z that are involved in 50- and 30-tRNA maturation, we ﬁnd ﬁve out of six subunits (Mtr4p; Dead-box family of ATP dependent helicase; ribosomal proteins Mrp1p, Mrpl3p, Mrpl35p and Mrpl40) of the RNA degradosome complex, as pre- viously characterized by others (34). The remaining second catalytic subunit of the RNA degradosome (30–50 exoribonuclease, Dss1p) was identiﬁed only once in the three LC-MS/MS experiments, which is consistent with its low level of cellular expression [only about 1000 mol- ecules/cell (51)]. The RNA degradosome is responsible for RNA turnover (34), but loss of function of any of its subunits also results in accumulation of RNA precursors with abnormal 50- and 30-termini, and stalled mitochon- drial translation (33,34). Finally, the presence of ﬁve further proteins involved in rRNA and mRNA processing and RNA modiﬁcation demonstrates a structural integra- tion of most mitochondrial RNA processing activities (Nop7p; involved in rRNA processing, Mrm1p; ribose methyltransferase of the mitochondrial 21S rRNA, Hsh155p; mRNA-binding protein, Ngl1p; putative endo- nuclease and Prp22p; DEAH-box RNA-dependent ATPase/ATP-dependent RNA helicase). That the RMT complex contains 22 ribosomal proteins of the large subunit and 13 of the small subunit is in agree- ment with the existing data that show an association of a highly puriﬁed RNA degradosome complex with four ribosomal proteins (Mrpl3p, Mrpl35p, Mrpl40 and Mrp1p) (34). Depending on the adopted experimental Figure 4. Association of mt-RNase P with 30-tRNA processing activity. The RMT complex is assayed for in vitro processing of 50-leader and 30-termini pre-tRNAproline. Assay conditions are given in ‘Materials and Methods’ section. The negative control (C–) is without addition of M1 RNA and the positive control is with M1 RNA (+M1). The RNA corresponding to 50-leader and 30-terminus pre-tRNAproline (148 nt), 50-leader pre-tRNAproline (117 nt), tRNAproline (78 nt), 50-leader (39 nt), and 30-trailer (31 nt) sequences are indicated. Activities of the combined mt-RNase P and 30-tRNA processing activity were found in fractions 36 and 37.