Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites.

Abstract

Abstract. Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix.Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy ,x,l% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to ~7 % of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.

Mitochondrial protein import: Specific recognition and membrane translocation of preproteins

“…In the first case, the translocation sites of the outer membrane were saturated with translocation intermediates spanning the two membranes. This treatment led to an import inhibition of other precursor proteins (Vestweber & Schatz, 1988;Rassow et al, 1989). However, import of some precursor proteins could be restored if the outer membrane of these mitochondria was opened (Hwang et al, 1989).…”

Contact sites between inner and outer membranes

“…Contact sites occupy about 7-15% of the mitochondrial outer membrane surface [7,18]. As far as it can be assessed by electron microscopy, the two membranes are not fused at contact sites.…”

“…Rather two intact bilayers are always visible that are separated by an optically non-dense structure. The distance from the cytosolic side of the outer membrane to the matrix side of the inner membrane is about 18 nm [18]. By saturation of contact sites with precursor proteins it was calculated that about 100-5000 translocation sites were present in one isolated mitochondrion [18,19].…”

“…The distance from the cytosolic side of the outer membrane to the matrix side of the inner membrane is about 18 nm [18]. By saturation of contact sites with precursor proteins it was calculated that about 100-5000 translocation sites were present in one isolated mitochondrion [18,19]. It is so far unclear as to whether the morphologically visible contact sites are part of a large and coherent network ('contact stripes') or whether multiple non-coherent contact sites exist.…”

“…Various methods were developed for the accumulation of precursor proteins in mitochondrial contact sites: import of precursor proteins at low temperature [9,15,20]; lowering the levels of ATP in the import reaction [21]; prebinding of specific antibodies to portions of the precursor proteins [9,20]; and, induction of a stable tertiary structure in a domain of a precursor protein [18,19,22]. The underlying principle of these methods is that a (usually carboxyl-terminal) portion of a precursor protein is either not or only partially unfolded and thus cannot be inserted into the mitochondrial membranes.…”