Mitochondria play a crucial role in many cellular processes such as energy production, apoptosis and fatty acid metabolism, and are essential organelles of almost all eukaryotic cells. They contain a functional genome, which is considered to be a relic from their free-living proteobacterial ancestors1
. However the mitochondrial genome contains only a few genes, encoding just eight proteins in Saccharomyces cerevisiae2
, whereas proteomic studies have revealed around 800 distinct mitochondrial proteins in this organism3
. Thus, the majority of mitochondrial proteins have to be imported from the cytosol.
Protein import into mitochondria is driven by a multi-component system, with components located in the cytosol, in both mitochondrial membranes, in the intermembrane space and in the mitochondrial matrix. Import occurs in several steps (for review, see4
). N-terminal transit peptides of mitochondrially targeted proteins are recognized by the receptors of the TOM-complex (Translocase of the Outer Membrane) Tom20, Tom22 and Tom705
and then transported through the outer membrane via the Tom40 protein channel6
. Outer membrane proteins with β-barrel topology (e.g. Tom40, porins) after passing through the protein-conducting channel are inserted by another outer membrane complex, SAM (Sorting and Assembly Machinery) (for review, see7
). The proteins that are targeted to other mitochondrial sub-compartments undergo further transport through the inner membrane, and, depending on their final destination, are recognized by one of the two inner membrane translocases: TIM22 or TIM23 (for review, see8,9
Although the components and mechanisms of the mitochondrial protein transport machinery are well understood10,11,12,13,14
, knowledge about the movement of these proteins in the mitochondrial membrane is scarce. Research in this direction is currently limited to a recent investigation of a fluorescently labeled Tom40-associated subunit of the TOM complex, Tom7 by Fluorescence Recovery After Photobleaching (FRAP)15
, a technique previously used for investigating protein diffusion in the mitochondrial matrix16,17,18,19
. Tom7 was observed to have strongly heterogeneous diffusive properties: the majority of the protein population is freely diffusive, with a diffusion coefficient along the mitochondrial axis in the outer membrane of 0.7 μm2
/s, whereas a minor sub-population (7%) is virtually immobile15
In the absence of direct experimental investigations of Tom40 movement in the mitochondrial membrane, it was suggested to diffuse freely in the outer mitochondrial membrane, based on the absence of patterns of Tom40 distribution in the outer mitochondrial membrane in electron microscopy investigations20,21
In this report we focus on the investigation of how the TOM complex diffuses as a unit: are the protein import pores anchored in the mitochondrial membrane or are they freely moving? Unfortunately, GFP-labeled Tom7 as previously used15
is not a reliable proxy for the investigations of the diffusive dynamics of the TOM complex. First, it is non-essential for mitochondrial functionality (Δtom7
yeast strain is viable, although it exhibits a slight reduction in growth at higher temperatures (37°C) on non-fermentative media22
). This makes it hard to assay for functionality of the Tom7 modified as a Tom7-GFP fusion, especially in the case of the transfection-based approach used by Sukhorukov et al.15
(see below). Second, Tom7 is known to associate with the TOM complex in a dynamic fashion23,24
, which makes it an even more unreliable readout for TOM diffusion. In contrast, Tom40, being a central component of the TOM complex, is essential for the mitochondrial functionality25
, and hence is an ideal labeling target for monitoring dynamics of the TOM complex.
A promising alternative for studying the diffusive behavior of TOM components by FRAP is Single Particle Tracking (SPT) fluorescence microscopy of GFP-tagged proteins. However powerful, this technique has one serious limitation: autofluorescence of the cytoplasm efficiently masks signal from a single GFP molecule, constituting a serious problem for imaging in yeast cytosol or deep within mammalian cells. Therefore this approach has been limited to the studies of bacteria or, in total internal reflection mode, outer mammalian membrane proteins26
. SPT analysis of the cytosolic diffusion is possible, but requires fluorophores that are considerably brighter than the currently available GFP variants, or the tethering of several GFP molecules to the target, e.g. labeling mRNA with a dozen GFPs for tracking in the yeast cytoplasm27
. Even though the SPT technique has revolutionized investigations of eukaryotic outer membrane proteins28,29
, due to the limitations discussed above, it has never been applied to mitochondrial targets.
The brunt of biochemical and in vivo
investigations of mitochondrial transport is carried out using Saccharomyces cerevisiae
as a model organism30,31,32
. The main advantage of using yeast for biochemical and in vivo
investigations is its high amenability to genetic manipulations. However, studies of the spatial distribution33
and diffusion dynamics15
of components of the TOM complex were performed in mammalian cells transfected with expression constructs for the fluorescently labeled proteins of interest. Even though mammalian cells have somewhat lower fluorescence background than yeast cells, they are much more complex in their genetic manipulations which necessitates usage of the transient expression from a transfection construct. This, however, leads to a heterogeneous protein population, where the fluorescently labeled protein of interest is expressed in the presence of non-labeled one, and in concentrations exceeding normal expression levels. Thus only a portion of the labeled protein may be engaged in functional interactions.
In this report we present an alternative approach to studying diffusional dynamics of individual yeast mitochondrial components. We constructed a S. cerevisiae
strain in which Tom40 is expressed under control of its natural promoter as a fusion with the GFP variant Dendra234
in the absence of unlabeled protein. Due to the high autofluorescence of the yeast cytoplasm, rather than imaging intact cells, we imaged isolated yeast mitochondria using a state-of-the-art SPT setup ()35
. While our assay inherently separates mitochondrial movement within the cell from movement of Tom40 in the mitochondrial membrane, it must be noted that effects of Tom40 interactions with cytoplasmic components such as transport events or subunit exchange are not taken into account in our model system in its current implementation.
Schematic diagram of the optical setup.
The analysis of individual Tom40 trajectories in intact mitochondria recorded with a frame rate of 5 ms reveals that the movement in the outer mitochondrial membrane is highly dynamic but confined in nature, suggesting anchoring of the TOM complex as a whole.