GDI-Freisetzungsmechanismen: Nukleotidaustausch zur Regulation der Interaktion von prenylierten Rab-Proteinen und GDI

Rab‐Proteine regulieren als molekulare Schalter eine Vielzahl von intrazellulären Transportprozessen. Diese Regulation erfolgt in zwei miteinander verschalteten Zyklen. Im ersten Zyklus wechseln die Rab‐Proteine zwischen einer membranständigen und einer cytoplasmatischen Lokalisation. Die Interaktion mit der Membran erfolgt durch die hydrophoben C‐terminalen Prenylanker der Rab‐Proteine, die – zur Erhöhung der Löslichkeit – im Cytoplasma von dem Protein GDI gebunden werden. Im zweiten Regulationszyklus werden die Rab‐Proteine aktiviert bzw. deaktiviert und so die Interaktion mit Effektorproteinen und die Aktivierung von Signalkaskaden reguliert. Da die Aktivierung durch Nukleotidaustausch zu GTP und die Deaktivierung der Rab‐Proteine durch Nukleotidhydrolyse in der Regel durch membranlokalisierte Proteine erfolgt, sind die zwei Regulationszyklen miteinander verschaltet. Die Aktivierung und Deaktivierung der Rab‐ Proteine wurde bis zum heutigen Zeitpunkt relativ detailliert charakterisiert, während wenige Daten zur Regulation der Rab‐Lokalisation vorliegen. DrrA, ein Protein aus dem Bakterium Legionella pneumophila, wurde als dual funktionaler Faktor identifiziert, der sowohl die Lokalisation durch Dissoziation des Rab:GDI‐Komplexes als auch die Aktivierung von Rab1 durch Nukleotidaustausch zu GTP beeinflussen kann (Machner und Isberg, 2007; Ingmundson et al., 2007). Diese Aktivitäten wurden in dieser Arbeit näher charakterisiert und es konnte gezeigt werden, dass es sich nicht um zwei separate Aktivitäten handelt. Die Charakterisierung der Nukleotidaustauschaktivität zeigte, dass DrrA ein sehr effektiver Nukleotidaustauschfaktor für Rab1 ist und es konnte gezeigt werden, dass diese Nukleotidaustauschaktivität die Basis für die in der Literatur beschriebene GDIFreisetzungsaktivität von DrrA ist. Aufgrund der Tatsache, dass die Affinität von GDI für GTPgebundenes Rab deutlich geringer ist als für GDP‐gebundenes Rab (Wu et al., 2010), kann DrrA durch Nukleotidaustausch verhindern, dass nach einer Dissoziation des Rab1:GDIKomplexes eine Reassoziation erfolgt. Hierdurch verschiebt DrrA das Gleichgewicht zwischen GDI‐gebundenem und freiem Rab‐Protein und bewirkt so indirekt eine vollständige Dissoziation des Rab1:GDI‐Komplexes. Um zu demonstrieren, dass dieser Freisetzungsmechanismus auf andere Rab‐Proteine und ihre Nukleotidaustauschfaktoren übertragbar ist, wurde diese Studie systematisch auf weitere Rab‐Proteine und ihre Nukleotidaustauschfaktoren erweitert. Der Nukleotidaustausch zu GTP nach der Dissoziation eines Rab:GDI‐Komplexes konnte bei allen untersuchten Rab‐Proteinen die Reassoziation der Komplexe inhibieren und so eine Freisetzung der Rab‐Proteine aus dem Komplex mit GDI bewirken. Diese Daten zeigen, dass der Regulationszyklus der Aktivierung von Rab‐Proteinen den Lokalisationszyklus prinzipiell beeinflussen kann, und somit die Lokalisation der Rab‐ Proteine durch ihre Aktivierung beeinflusst werden kann

Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor

Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane extraction/delivery and cytosolic distribution of Rabs is mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement of GDI from Rab:GDI complexes. These activities have been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI

Reversible phosphocholination of Rab proteins by Legionella pneumophila effector proteins

The Legionella pneumophila protein AnkX that is injected into infected cells by a Type IV secretion system transfers a phosphocholine group from CDP-choline to a serine in the Rab1 and Rab35 GTPase Switch II regions. We show here that the consequences of phosphocholination on the interaction of Rab1/Rab35 with various partner proteins are quite distinct. Activation of phosphocholinated Rabs by GTP/GDP exchange factors (GEFs) and binding to the GDP dissociation inhibitor (GDI) are strongly inhibited, whereas deactivation by GTPase activating proteins (GAPs) and interactions with Rab-effector proteins (such as LidA and MICAL-3) are only slightly inhibited. We show that the Legionella protein lpg0696 has the ability to remove the phosphocholine group from Rab1. We present a model in which the action of AnkX occurs as an alternative to GTP/GDP exchange, stabilizing phosphocholinated Rabs in membranes in the GDP form because of loss of GDI binding ability, preventing interactions with cellular GTPase effectors, which require the GTP-bound form. Generation of the GTP form of phosphocholinated Rab proteins cannot occur due to loss of interaction with cellular GEFs

Image_2_The Loss of Expression of a Single Type 3 Effector (CT622) Strongly Reduces Chlamydia trachomatis Infectivity and Growth.TIF

<p>Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the Chlamydiaceae, in the absence of which most bacteria failed to establish a successful infection. CT622 is abundant in the infectious form of the bacteria, in which it associates with CT635, a putative novel chaperone protein. We show that CT622 is translocated into the host cytoplasm via type three secretion throughout the developmental cycle of the bacteria. Two separate domains of roughly equal size have been identified within CT622 and a 1.9 Å crystal structure of the C-terminal domain has been determined. Genetic disruption of ct622 expression resulted in a strong bacterial growth defect, which was due to deficiencies in proliferation and in the generation of infectious bacteria. Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. They reveal that genetic disruption of a single effector can deeply affect bacterial fitness.</p

Table_2_The Loss of Expression of a Single Type 3 Effector (CT622) Strongly Reduces Chlamydia trachomatis Infectivity and Growth.PDF

<p>Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the Chlamydiaceae, in the absence of which most bacteria failed to establish a successful infection. CT622 is abundant in the infectious form of the bacteria, in which it associates with CT635, a putative novel chaperone protein. We show that CT622 is translocated into the host cytoplasm via type three secretion throughout the developmental cycle of the bacteria. Two separate domains of roughly equal size have been identified within CT622 and a 1.9 Å crystal structure of the C-terminal domain has been determined. Genetic disruption of ct622 expression resulted in a strong bacterial growth defect, which was due to deficiencies in proliferation and in the generation of infectious bacteria. Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. They reveal that genetic disruption of a single effector can deeply affect bacterial fitness.</p