Prerequisite For Translocation Research Articles

Aggregation of Oligoarginines at Phospholipid Membranes: Molecular Dynamics Simulations, Time-Dependent Fluorescence Shift, and Biomimetic Colorimetric Assays

A time-dependent fluorescence shift method, biomimetic colorimetric assays, and molecular dynamics simulations have been performed in search of explanations why arginine rich peptides with intermediate lengths of about 10 amino acids translocate well through cellular membranes, while analogous lysine rich peptides do not. First, we demonstrate that an important factor for efficient peptide adsorption, as the first prerequisite for translocation across the membrane, is the presence of negatively charged phospholipids in the bilayer. Second, we observe a strong tendency of adsorbed arginine (but not lysine) containing peptides to aggregate at the bilayer surface. We suggest that this aggregation of oligoarginines leads to partial disruption of the bilayer integrity due to the accumulated large positive charge at its surface, which increases membrane-surface interactions due to the increased effective charge of the aggregates. As a result, membrane penetration and translocation of medium length oligoarginines becomes facilitated in comparison to single arginine and very long polyarginines, as well as to lysine containing peptides.

Acid-triggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure

Exposure to low endosomal pH during internalization of Pseudomonas exotoxin A (PE) triggers membrane insertion of its translocation domain. This process is a prerequisite for PE translocation to the cytosol where it inactivates protein synthesis. Although hydrophobic helices enable membrane insertion of related bacterial toxins such as diphtheria toxin, the PE translocation domain is devoid of hydrophobic stretches and the structural features triggering acid-induced membrane insertion of PE are not known. Here we have identified a molecular device that enables PE membrane insertion. This process is promoted by exposure of a key tryptophan residue. At neutral pH, this Trp is buried in a hydrophobic pocket closed by the smallest alpha-helix of the translocation domain. Upon acidification, protonation of the Asp that is the N-cap residue of the helix leads to its destabilization, enabling Trp side chain insertion into the endosome membrane. This tryptophan-based membrane insertion system is surprisingly similar to the membrane-anchoring mechanism of human annexin-V and could be used by other proteins as well.

Evidence That Translocation of the Proteinase Precedes Its Acylation in the Serpin Inhibition Pathway

The inhibition of proteinases by serpins involves cleavage of the serpin, acylation, and translocation of the proteinase. To see whether acylation precedes or follows translocation, we have investigated the pH dependence of the interaction of fluorescein isothiocyanate-elastase with rhodamine alpha(1)-proteinase inhibitor (alpha(1)PI) using two independent methods: (i) kinetics of fluorescence energy transfer which yields k(2,f), the rate constant for the fluorescently detected decay of the Michaelis-type complex (Mellet, P., Boudier, C., Mély, Y., and Bieth, J. G. (1998) J. Biol. Chem. 273, 9119-9123); (ii) kinetics of elastase-catalyzed hydrolysis of a substrate in the presence of alpha(1)PI, which yields k(2,e), the rate constant for the conversion of the Michaelis-type complex into irreversibly inhibited elastase. Both rate constants were found to be pH-independent and close to each other, indicating that acylation, a pH-dependent phenomenon, does not govern the decay of the Michaelis-type complex and, therefore, follows translocation. On the other hand, anhydro-elastase reacts with alpha(1)PI to form a Michaelis-type complex that translocates into a second complex with a rate constant close to that measured with active elastase, confirming that acylation is not a prerequisite for translocation. Moreover, the anhydro-elastase-alpha(1)PI complex was found to be thermodynamically reversible, suggesting that translocation of active elastase might also be reversible. We propose that serpins form a Michaelis-type complex EI(M), which reversibly translocates into EI(tr) whose acylation yields the irreversible complex EI(ac). [see text]

Mitogen-activated protein kinase translocates to the nucleus during ischaemia and is activated during reperfusion.

Growth factors and various cellular stresses are known to activate mitogen-activated protein (MAP) kinase, which plays a role in conveying signals from the cytosol to the nucleus. The phosphorylation of MAP kinase is thought to be a prerequisite for translocation. Here, we investigate the translocation and activation of MAP kinase during ischaemia and reperfusion in perfused rat heart. Ischaemia (0-40 min) induces the translocation of MAP kinase from the cytosol fraction to the nuclear fraction. Immunohistochemical observation shows that MAP kinase staining in the nucleus is enhanced after ischaemia for 40 min. Unexpectedly, tyrosine phosphorylation of MAP kinase is unchanged in the nuclear fraction during ischaemia, indicating that unphosphorylated MAP kinase translocates from the cytosol to the nucleus. During reperfusion (0-30 min), after ischaemia for 20 min, tyrosine phosphorylation of MAP kinase in the nuclear fraction is increased with a peak at 10 min of reperfusion. The activation is confirmed by MAP kinase activity with similar kinetics to the tyrosine phosphorylation. However, the amount of MAP kinase in the fraction is almost constant during reperfusion for 10 min. Although an upstream kinase for MAP kinase, MAP kinase/extracellular signal-regulated kinase kinase (MEK)-1, remains in the cytosol throughout ischaemia and reperfusion, MEK-2, another upstream kinase for MAP kinase, is constantly present in the nucleus as well as in the cytoplasm, based on analyses by fractionation and immunohistochemistry. Furthermore, MEK-2 activity in the nuclear fraction is rapidly increased during post-ischaemic reperfusion. These findings demonstrate that nuclear MAP kinase is activated by tyrosine phosphorylation during reperfusion, probably by MEK-2.

Translocation of Full-length Pseudomonas Exotoxin from Endosomes Is Driven by ATP Hydrolysis but Requires Prior Exposure to Acidic pH

We attached human transferrin to Pseudomonas exotoxin A (PE) to specifically localize this toxin to the endosomal compartment and study its translocation from purified endosomes using a cell-free assay. Transferrin was linked to PE via a disulfide bond. Chemical derivatization inactivated the PE cell-binding domain, and transferrin-PE was found to be endocytosed via the transferrin receptor only. Transferrin was also conjugated to a truncated PE with no receptor-binding domain (PE46). After labeling mouse lymphocytes with radiolabeled transferrin-PE or transferrin-PE46 and endosome isolation, selective translocation of the full-sized toxin portion of the conjugate was observed in a cell-free system. This translocation was strictly dependent upon ATP hydrolysis and was not affected when the acidity of the endosome lumen was neutralized using weak bases, protonophores, or bafilomycin A1. Nevertheless, when present during cell labeling, inhibitors of endosome acidification prevented PE from acquiring translocation competence. Similar inhibition was observed when endocytosis was performed in the presence of brefeldin A, a drug known to interfere with the delivery of endocytic tracers to acidic endosomes. Our data indicate that full-length PE can be transferred to the cytosol directly from endosomes during intoxication by PE conjugates and that, although exposure to acidic pH is a prerequisite for translocation, ATP hydrolysis directly provides the energy required for PE translocation.