Kinetic Studies on the Transport of Cytoplasmically Synthesized Proteins into the Mitochondria in Intact Cells of Neurospora crassa

Abstract

The transport of cytoplasmically synthesized mitochondrial proteins was investigated in whole cells of Neurospora crassa, using dual labelling and immunological techniques.In pulse and pulse-chase labelling experiments the mitochondrial proteins accumulate label. The appearance of label in mitochondrial protein shows a lag relative to total cellular protein, ribosomal, microsomal and cytosolic proteins.The delayed appearance of label was also found in immunoprecipitated mitochondrial matrix proteins, mitochondrial ribosomal proteins, mitochondrial carboxyatractyloside-binding protein and cytochrome c. Individual mitochondrial proteins exhibit different labelling kinetics.Cycloheximide inhibition of translation does not prevent import of proteins into the mitochondria. Mitochondria1 matrix proteins labelled in pulse and pulse-chase experiments can first be detected in the cytosol fraction and subsequently in the mitochondria. The cytosol matrix proteins and those in the mitochondria show a precursor-product type relationship.The results suggest that newly synthesized mitochondrial proteins exist in an extra-mitochondria1 pool from which they are imported into the mitochondria.The majority of mitochondrial proteins are coded for by nuclear genes and are synthesi~ed on cytoplasmic ribosomes. It has been estimated that some 85 -95 % of the total mitochondrial protein originates from cytoplasmic protein synthesis [l]. These proteins must subsequently be transported to their functional locations in the mitochondrion. A similar situation exists in the formation of chloroplasts in plant cells where the majority of the chloroplast proteins originate in the cytoplasm [2].Despite the obvious importance of the transport process little is known about the selectivity which allows only mitochondrial proteins access to mitochondria and about the transport mechanism. In recent years a mechanism was proposed which integrates synthesis and transport into a simple unified mechanism [ 3 -61. According to this attractive hypothesis, mitochondrial proteins are translated by cytoplasmic ribosomes which are bound to the outer membrane of the mitochondria. Nascent polypeptides are discharged in a vectorial manner into the matrix, or membranes, in a manner analogous to the mechanism proposed for protein transport in exocrine cells [7]. The evidence for the general existence of such a mechanism is equivocal and many questions in relation to how such a system could function remain unanswered.We have studied the kinetics of synthesis of mitochondrial proteins and their subsequent appearance in the mitochondria in intact cells of Neurospora cru,ssu. The results of our studies suggest that extramitochondrial pools of mitochondrial proteins exist. It is furthermore shown that newly synthesized mitochondrial proteins can be detected outside the mitochondrial fraction. Our data are not readily compatible with a mechanism in which synthesis and transport are functionally linked. The existence of cytoplasmic ribosomes bound to outer membranes of mi...

Transport of Cytoplasmically Synthesized Proteins into the Mitochondria in a Cell Free System from Neurospora crassa

“…The nature of the immunoprecipitated matrix proteins was investigated by subjecting the immunoprecipitates to dodecylsulfate gel electrophoresis. There was not exact coincidence between the 35S and 3H radioactivity peaks, but the patterns obtained match those obtained in vivo after short periods of labelling [1,2].…”

Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space

“…The kinetics of export are slower than those of import (Hart1 et al, 1987). The uptake of proteins into mitochondria in vivo is a very rapid process (Hallermayer et al, 1977) occurring with kinetics comparable to those observed with import of artificially unfolded precursor proteins in vitro. Under physiological conditions, proteins destined for export may not get immediate access to an export site in the inner membrane during import.…”

Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts

“…This approach was a real Copernican revolution for mitochondrial research, allowing the isolation of pure organelles with high yields. As a practical consequence, in the subsequent 20 years, we saw such amazing discoveries: the mechanism of energy conservation 4 ; the identification of mitochondrial DNA 5,6 and of import of mitochondrial precursor proteins 7 ; the definition of mitochondrial ultrastructure, with the development of the so-called ''Palade's model'' 8 ; and last but not least, the discovery of inner mitochondrial membrane channels 9 .…”