Journal of Molecular Biology
ReviewInsertion of Hydrophobic Membrane Proteins into the Inner Mitochondrial Membrane—A Guided Tour
Introduction
Mitochondria of different species have been estimated to harbor between 500 to 2000 different proteins distributed over four sub-compartments: the outer membrane, intermembrane space, inner membrane and matrix.1 This large number of proteins represents a reflection of the numerous metabolic functions housed within mitochondria. While there is no doubt that mitochondria fulfill vital tasks for any given cell, it is surprising to see how many and which of these functions are dispensable in lower eukaryotes, such as the yeast Saccharomyces cerevisiae. The completion of the yeast genome and systematic gene deletion analyses provide an overview into which mitochondrial proteins are essential and to which functional category they belong. Surprisingly only a few mitochondrial proteins are essential for cell viability and these proteins are involved in protein transport, protein folding, or Fe/S cluster biogenesis while all other strictly mitochondrial proteins seem to be dispensable. The most likely explanation for this finding is that any disturbances in these three tasks affects a large variety of proteins involved in different cellular functions, thus causing a synthetic phenotype by leading to inactive proteins or abrogating their proper sorting (see Appendix A for details). In contrast, e.g. all complexes of the respiratory chain of yeast mitochondria are dispensable on fermentable growth medium.
In the yeast S. cerevisiae only eight and in humans just 13 mitochondrial proteins are encoded by the organellar genome. Therefore, about 99% of the mitochondrial proteins are nuclear encoded and have to be imported into the organelle from the cytosol. Even though a number of genomes from various organisms have been sequenced, for all of them it remains unknown which of the herein encoded proteins will eventually be localized in mitochondria. This lack of valuable information is mainly based on the difficulty to identify all mitochondrial proteins in silico. Although a number of algorithms have been designed to predict mitochondrial targeting signals they only work with an accuracy of about 60%2 and can detect only those proteins that utilize an amino-terminal pre-sequence. Other targeting information, e.g. those of proteins with internal targeting signals, cannot be predicted at all. The reason for this significant gap is the fact that internal signals have not yet been characterized well enough to allow a computer-based prediction. Therefore, it remains a challenge to the field of mitochondrial protein transport to define such signals on a molecular level and within as many cargo proteins as possible in order to build a statistical basis for a more general signal concept.
Aside from outer membrane and intermembrane space proteins, many proteins with internal targeting signals are polytopic membrane proteins of the inner mitochondrial membrane. Most of these proteins are involved in metabolite transport (carrier proteins) or even protein transport (Tim23, Tim22). The fact that all of them are integral membrane proteins with more than one transmembrane span, which have to be imported post-translationally into mitochondria, creates a technical problem for any given cell. Since mitochondria can only import unfolded or loosely folded proteins, the cell has to ensure that on the route from the cytosol to the inner membrane the precursor proteins remain in a transport-competent state, typically a loosely folded conformation. In addition, the extensive hydrophobic regions that will eventually form the transmembrane spans have to be shielded in aqueous environments, such as those found in the cytoplasm and the intermembrane space, in order to prevent protein aggregation. To tackle this problem, a number of chaperones in these compartments act as molecular “tour guides” for the polytopic membrane proteins, helping to ensure appropriate folding while preventing aggregation and assisting in recognition and transfer of cargo proteins across membranes. Here, we want to focus on this guided trip for hydrophobic precursor proteins to the inner membrane and present recent findings on how their targeting and assembly is achieved.
Section snippets
Targeting signals of mitochondrial proteins
In principle, one can discriminate between two types of signals that direct proteins to mitochondria: amino-terminal signals and internal signals.
Most mitochondrial matrix proteins utilize an amino-terminal signal, which is termed a pre-sequence. The pre-sequence consists of the first 10–80 amino acid residues of the protein and is usually cleaved off by a protease found in the mitochondrial matrix (matrix processing peptidase, MPP) during or after import.3 A characteristic feature of
The translocase of the outer mitochondrial membrane–TOM complex
Mitochondrial targeting signals are recognized by receptor molecules on the outer surface of mitochondria. Three integral outer membrane proteins have thus far been identified as receptors and characterized with regard to their specificities: Tom20,20., 21., 22. Tom22,23., 24., 25., 26., 27. and Tom70.28., 29., 30., 31., 32., 33.
Tom20 and Tom70 are considered to be the primary receptors for the two classes of precursor proteins. Pre-proteins with a pre-sequence are initially bound to Tom20 via
Transport of pre-sequence-containing proteins to the mitochondrial matrix
Since this paper is mainly dedicated to the transport of hydrophobic proteins to the mitochondrial inner membrane we will only briefly summarize some aspects of how pre-sequence-containing proteins are transported to the matrix. More detailed descriptions of this transport pathway can be found in other reviews.51., 52., 53., 54.
Pre-sequence-containing proteins are initially recognized by Tom20,20., 21. which binds to the hydrophobic face of the α-helix formed by the pre-sequence.7., 34. The
Driving forces for transport through the pre-sequence translocase
Besides its involvement in regulation of the translocase, the membrane potential also represents a driving force for the transfer of proteins across the inner membrane.67., 68., 69. The generally accepted idea of how a driving force on the pre-protein can be generated by the membrane potential is that it creates an electrophoretic effect on the positively charged pre-sequence.67., 70. Accordingly, the membrane potential mediates the initial transfer of the pre-protein through the Tim23 channel.
Transport of hydrophobic proteins to mitochondria is a multi-stage process
The transport of proteins with several membrane spanning segments to mitochondria is especially demanding for the cell. The precursor proteins have to be maintained in a translocation-competent state and the multiple hydrophobic stretches have to be shielded against the aqueous environment of the cytosol in order to protect them from aggregation. It seems likely that molecular chaperones are involved in this task.91., 92., 93., 94., 95. Nevertheless, it is still not clear how they achieve this
Defining the components that mediate the translocation of polytopic membrane proteins across the outer membrane
As mentioned above, polytopic membrane proteins are initially recognized on the outer surface of mitochondria by the cytosol-exposed domain of Tom70.31., 33., 100., 101. In addition to Tom70 it appears that Tom20 is also capable of recognizing carrier proteins. Upon deletion of TOM70, import of AAC can be mediated with low efficiency by Tom20.32 Moreover, the cytosolic domain of Tom20 is able to bind to the phosphate carrier in vitro.34 However, under physiological conditions, Tom70 acts as the
The protein insertion complex (TIM22 complex) of the inner mitochondrial membrane
Tim12 represents a protein with high sequence similarity to Tim9 and Tim10, but in contrast to these is stably associated with the mitochondrial inner membrane.103., 106. Like Tim9 and Tim10, Tim12 is also essential for cell viability and thus performs an important function in protein translocation. Tim12 is part of the protein insertion complex of 300 kDa in the inner membrane (also called the TIM22 complex) that includes the integral membrane proteins Tim22, Tim54 and Tim18. Small amounts of
The Tim8–Tim13 complex
Not only metabolite carriers utilize the TIM22 complex for transport into the inner membrane but also some subunits of the translocases themselves. All Tom and Tim proteins are nuclear encoded and must be imported into mitochondria. The precursor of Tim17, Tim23, and Tim22 are synthesized without pre-sequence and possess multiple hydrophobic segments. It is clear that the precursors of Tim22 and Tim23 are transported via the TIM22 complex,115., 122. whether the same holds true for Tim17 is
Perspectives
As we continue analyzing how proteins are transported to and sorted within mitochondria, we have to face the fact that we have just started to peek at the molecular mechanisms underlying these processes. Only recently a novel integral component of the protein insertion complex has been identified113., 114. and we have to assume that for this and other complexes involved in protein transport new components still await their discovery (see also Appendix A).
Besides the TOM complex, no other
Acknowledgements
We are grateful to A. E. Frazier and N. Wiedemann for critical comments on the manuscript. Work of the authors' laboratory was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich 388 Freiburg and the Fonds der Chemischen Industrie/BMBF.
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