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The association between ribosomes and the pore proteins at the endoplasmic reticulum membrane is important to co-translational translocation. To determine if a similar association occurs between the ribosome and mitochondrial membrane protein(s) during protein import in higher eukaryotes, we examined ribosome-mitochondria binding. By using spectral measurements, analysis of mitochondrial associated RNA, and electron microscopy, we demonstrated that ribosomes stably bind to purified rat liver mitochondria in vitro. Binding of ribosomes to mitochondria was markedly reduced by GTP and nearly abolished by the non-hydrolyzable GTP analogue, guanosine-5-[thio]-triphosphate (GTP␥S), but was only modestly reduced by GDP or ATP and unaffected by CTP. The initial rate of GTP hydrolysis by mitochondria was increased by ribosomes, whereas the rate of ATP hydrolysis by mitochondria was not affected. Ribosomes programmed with mRNA for 92 amino acids of the N terminus of mitochondrial malate dehydrogenase bound to mitochondria, but unlike unprogrammed rat liver ribosomes, neither GTP nor GDP disrupted binding; however, GTP␥S did. These data show that receptors specific for ribosomes are present on the mitochondrial membrane, and a GTP-dependent process mediates this binding. The presence of a nascent chain alters these binding characteristics. These findings support the hypothesis that a co-translational translocation pathway exists for import of proteins into mitochondria.Proteins can cross membranes by either post-translational or co-translational translocation. Based on in vitro observations, proteins targeted to the mitochondria are thought to be completely synthesized in the cytoplasm and cross the mitochondrial membrane(s) post-translationally (1). However, evidence consistent with a co-translational translocation pathway for mitochondrial protein import has been reported. For example, 1) the surface of mitochondria isolated from cycloheximidetreated yeast cells is observed to be studded with polysomes (2); 2) the number of bound polysomes is dependent on the metabolic state of the cells from which the mitochondria are isolated (3); 3) ribosomes are bound to the mitochondria at the contact sites (4); and 4) the mRNA of polysomes that co-isolate with mitochondria is enriched in messages for mitochondrial proteins (5, 6). In addition, both in vitro and in vivo, mitochondrial protein import can be instantaneously disrupted by cycloheximide-induced translation arrest indicating that no detectable pool of full-length precursor exists in the cytosol and that translation and import are tightly coupled (7-9). Furthermore, methotrexate, which inhibits post-translational import of cytochrome oxidase subunit IV-dihydrofolate reductase by preventing its unfolding, does not inhibit cytochrome oxidase subunit IV-dihydrofolate reductase import in vivo (9). This indicates that co-translational translocation may eliminate the need for precursor proteins to be maintained in a translocation-competent state by chaperones in the cytoplasm.Al...

The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), hemoglobin (Hb)], and two pepsin resistant proteins [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to assessing safety of proteins.