Organization and Function of the Small Tim Complexes Acting along the Import Pathway of Metabolite Carriers into Mammalian Mitochondria

Abstract

Tim9, Tim10a, and Tim10b are members of the family of small Tim proteins located in the intermembrane space of mammalian mitochondria. In yeast, members of this family act along the TIM22 import pathway during import of metabolite carriers and other integral inner membrane proteins. Here, we show that the human small proteins form two distinct hetero-oligomeric complexes. A 70-kDa complex that contains Tim9 and Tim10a and a Tim9-10a-10b that is part of a higher molecular weight assembly of 450 kDa. This distribution among two complexes suggests Tim10b to be the functional homologue of yeast Tim12. Both human complexes are tightly associated with the inner membrane and, compared with yeast, soluble 70-kDa complexes appear to be completely absent in the intermembrane space. Thus, the function of soluble 70-kDa complexes as trans-site receptors for incoming carrier proteins is not conserved from lower to higher eukaryotes. During import, the small Tim complexes directly interact with human adenine nucleotide translocator (ANT) in transit in a metal-dependent manner. For insertion of carrier preproteins into the inner membrane, the human small Tim proteins directly interact with human Tim22, the putative insertion pore of the TIM22 translocase. However, in contrast to yeast, only a small fraction of Tim9-Tim10a-Tim10b complex is in a stable association with Tim22. We conclude that different mechanisms and specific requirements for import and insertion of mammalian carrier preproteins have evolved in higher eukaryotes.Most mitochondrial proteins are encoded by nuclear genes, translated at cytosolic ribosomes, and imported to their final destination in the mitochondrion. Import of nuclear-encoded mitochondrial preproteins is a multistep process mediated by a common translocase in the outer membrane, the TOM 1 complex, and two distinct translocases in the inner membrane, named TIM23 and TIM22 complex (1-4). The TOM complex is used by all known nuclear-encoded precursors and translocates them into and across the outer membrane. The TIM23 and the TIM22 complex show different substrate specificities and energy requirements. Presequence-containing proteins are translocated across the inner membrane into the matrix via the TIM23 complex; whereas polytopic inner membrane proteins without presequences but with internal targeting signals use the TIM22 complex. The mitochondrial import pathways are evolutionary conserved and many components of the mitochondrial import machineries characterized in the fungal system have counterparts in mammalian mitochondria (5). The TIM23 complex consists of the integral membrane components Tim23, Tim17, and Tim50. Tim23 and Tim17 constitute the protein-conducting channel and act in cooperation to form a receptor for presequences in the intermembrane space (6 -8). Tim50 exposing its large C-terminal domain to the intermembrane space was proposed to facilitate the transfer of precursor proteins to the translocation channel (9 -11). Translocation across the inner membrane is initially driven by t...

Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

“…Although the evolutionary conserved FXC1 encoded protein has not been directly associated with any human diseases, a loss of function mutation in a related component of the mitochondrial hetero-oligomeric translocase complex, TIMM8A, has been linked with the neurodegenerative disorder, Mohr-Tranebjaerg Syndrome [53]. The extensive protein-protein interactions occurring in the inner mitochondrial membrane translocase complex suggest that variation in FXC1 protein sequence caused by polymorphisms in its poly-Q region may have potential for impacting mitochondrial function [55]. Alternatively, the positioning of the poly-Q tract near the N-terminus of this small protein may have insulated its primary functions from the influence of polymorphic variation.…”

Mitochondrial protein translocases for survival and wellbeing

“…2C), this function is fulfilled by the metazoan‐specific TIMM10B (similar to yeast Tim9 and human TIMM9) [70]. TIMM10B forms a complex with TIMM9 and TIMM10, also referred to as Tim10a, and is present in the complex with TIMM22 [69]. TIMM10 and TIMM9 also form a separate complex without TIMM10B [69].…”

Functional and Mutational Characterization of Human MIA40 Acting During Import into the Mitochondrial Intermembrane Space

“…Peptide antibodies against human DDP1, TIM13, TIM10A, and TIM22 were raised as described. 25,26 Antibodies against human TIM23, protein disulfide isomerase, cytochrome c (CYC) and HSP70 were purchased from BD Biosciences; and b-actin antibodies were from Sigma. Western blot analyses using rabbit and mouse antibodies were performed as described.…”

“…Albeit a heterologous system, it has proven very useful for studying the biogenesis of mitochondrial proteins. 25,26 Mutant MIA40 constructs were generated using the megaprime protocol and expressed in yeast. Mitochondria were prepared from yeast transformants and assessed for MIA40 levels.…”