Protein folding causes an arrest of preprotein translocation into mitochondria in vivo.

Abstract

Abstract. With vital yeast cells, a hybrid protein consisting of the amino-terminal third of the precursor to cytochrome b2 and of the entire dihydrofolate reductase was arrested on the import pathway into mitochondria . Accumulation of the protein in the mitochondrial membranes was achieved by inducing a stable tertiary structure of the dihydrofolate reductase domain . Thereby, three salient features of mitochondrial protein up-ROTEIN translocation across biological membranes is a key step in the biogenesis of cell organelles (Wickner and Lodish, 1985). The basic problem ofhow cy tosolically synthesized precursor proteins traverse organelle membranes was studied predominantly in cell-free systems. These systems consisted of isolated organelles, such as microsomes (ER), mitochondria, chloroplasts or peroxisomes, and precursor proteins that were synthesized in vitro or expressed in Escherichia coli and subsequently purified. With regard to mitochondria, the following steps ofprotein import have been established (for reviews see Attardi and Schatz, 1988 ;Hard and Neupert, 1990; Pfanner and Neupert,1990) . Nuclear-encoded precursor proteins are synthesized on cytosolic polysomes and targeted to specific receptors on the outer mitochondrial membrane. A loosely folded ("unfolded") conformation of a precursor protein is a prerequisite for its translocation across the mitochondrial membranes (Schleyer and Neupert, 1985;Eilers and Schatz, 1986; Chenand Douglas, 1987a) . Most precursors are translocated through contact sites between outer and inner mitochondrial membranes (translocation contact sites) . The membrane potential across the inner membrane is required for the initial entrance of a precursor into this membrane, while the completion of translocation does not depend on A0. During or after membrane translocation, the amino-terminal targeting sequence (presequence) is proteolytically cleaved off by the processing peptidase in the mitochondrial matrix .Though posttranslational translocation of unfolded precursor proteins through mitochondrial contact sites is well established in the isolated system, little is known about protein transport into mitochondria in intact cells . In early studies performed at low temperature, cytoplasmic pools of mitochondrial precursor proteins were found in Neurospora

Import of cytochrome b2 to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP

“…Finally, some precursors will dissociate from the chaperones and fold quite rapidly, but will unfold again during subsequent translocation (Skerjanc et al, 1990). Mitochondrial precursors that contain either a DHFR moiety or the cytochrome b2 heme-binding domain probably belong in this last category (Eilers & Schatz, 1986;Pfanner et al, 1990;Wienhues et al, 1991;this study).…”

Transport of proteins across mitochondrial membranes

“…How does the IM remain sealed for protons when a polypeptide is crossing it? We have observed that no disturbance of the proton gradient occurs even when in a preprotein is accumulated in intact yeast cells in a membrane-spanning fashion to such a degree that the majority of import sites are occupied [70]. At the present time one can only speculate that the translocation pore in the IM is constructed in such a manner that it forms a "seal" around the chain in transit.…”

“…Morphological contact sites were described several years ago [21]. Preproteins in transit were localized to these sites by electron microscopy after binding of antibodies and protein A-gold [56,59,70]. Thus, the question arose as to whether the two translocation systems form a stable complex, or whether the nature of their interaction is more dynamic.…”

“…Intermediates have been accumulated whose N-terminus had reached the matrix space (and was processed by mitochondrial processing peptidase, MPP) whereas C-terminal parts of the preprotein were still exposed to the cytosolic compartment [56]. A number of conditions lead to arrest of translocation in such a fashion, for example, import at lower temperature, reduction of the ATP level in the extramitochondrial space, and presence of a tightly folded domain at the C-terminus of a preprotein [7,13,50,70]. The common theme of these different conditions is that they disfavor unfolding of preproteins in the cytosol.…”

Mitochondrial protein import: Specific recognition and membrane translocation of preproteins

“…Import into mitochondria can occur in a post-translational manner in vitro (Korb & Neupert, 1978;Maccecchini, Rudin & Schatz, 1979) and in vivo (Hallermayer, Zimmerman & Neupert, 1977;Schatz, 1979;Wienhues et al, 1991), showing that transport is mechanistically independent of elongation. Therefore, polypeptide chain elongation cannot be the driving force for mitochondrial protein import.…”