Podcasts about arf1

The protein GMAP-210 (Golgi Microtubule Associated Protein of 210 kDa) is a long coiled-coil protein, which localises to the Golgi apparatus. It is part of the loosely defined protein group of the golgins, which are involved in establishing the Golgi morphology and in vesicular trafficking around the Golgi. By using biochemical, cell biological and molecular biological methods GMAP-210 was examined in regards to its Golgi targeting capability, its interaction partners and its function in establishing Golgi morphology and positioning. In vitro and in vivo experiments showed that GMAP-210 targets to the Golgi via its C-terminal GRAB domain. Its proposed interaction with Arf1, however, could not be definitely determined, although there is strong evidence for it. Arf1 binding to the GRAB domain was hindered in the full-length protein, but not with short C-terminal fragments containing the minimal GRAB domain. This implies that additional factors are needed for GMAP-210 Golgi binding. A yeast 2-hybrid screen of the entire family of small Rab GTPases identified the Golgi and ER localised Rab1 as a novel interaction partner of GMAP-210. GMAP-210 also labels vesicular tubular structures in the cell, which partially overlap with COPII and ERGIC53, components of the early secretory pathway. This gives additional evidence that GMAP- 210 is involved in ER to Golgi transport. Trafficking of a model substrate, the vesicular stomatitis virus G-protein (VSV-G), however, was not impaired in the absence of GMAP- 210. This indicates that GMAP-210 functions only in specialised transport pathways. Knockdown of GMAP-210 in HeLa L cells by siRNA changed the Golgi morphology and the Golgi fragmented into a cluster of vesicles. Its overexpression caused the Golgi to grow long tubular structures. Both effects on morphology could only be observed in HeLa L cells, not in hTERT-RPE1 cells. As direct interaction with microtubules or γ-tubulin could not be detected, and GMAP-210 is therefore unlikely to affect Golgi morphology by directly perturbing microtubule function. GMAP-210 knockdown by siRNA also showed its interaction with the intraflagellar transport protein IFT20. This protein lost its Golgi localisation when GMAP-210 was depleted. Both proteins interacted directly. GMAP-210, however, was not involved in primary cilium formation in hTERT-RPE1 cells and loss of IFT20 from the Golgi did not impair formation of the cilium, proposing that the Golgi pool of IFT20 had a function apart from intraflagellar transport and formation of the primary cilium. These results set GMAP-210 apart from the archetypal golgins GM130 and p115 and indicate that GMAP-210 is involved in a highly specialised transport pathway, which could nevertheless influence the morphology of the Golgi apparatus in certain cell types.

The reaction product of phospholipase D (PLD), phosphatidic acid (PA), was found to stimulate phosphatidylinositol-4-phosphate-5-kinase (PIP-5-kinase) activity in vitro. In the present study, we have examined wether PLD affects the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) by PIP-5-kinase. Overexpression of PLD isoforms in HEK-293 cells led to an increase in PIP-5-kinase activity and to elevated PIP2 levels in intact cells. As both PLD and PIP-5-kinase are stimulated by the GTPases Arf1 and RhoA, we investigated in the following, if PLD is involved in the regulation of PIP2 synthesis by these GTPases. Both PLD1- and PLD2-induced PIP2 synthesis was completely blocked by coexpression of catalytically inactive Arf1 T31N. Reversely, the effect of constitutive active Arf1 Q71L was fully inhibited by catalytically inactive PLD constructs. Whereas the effects of Arf1 Q71L and wild-type PLD2 were additive, coexpression of Arf1 Q71L with wild-type PLD1 led to a synergistic increase in PIP-5-kinase activity. Previously, we have shown that RhoA regulates the activity of PLD and PIP-5-kinase by its downstream effector Rho-kinase. Expression of small amounts of inactive PLD1, but not of PLD2, nearly completely abolished Rho-kinase-stimulated PIP-5-kinase activity. Also expression of a non-phosphorylatable mutant of cofilin, which participates in the signalling cascade from RhoA via Rho-kinase and LIM-kinase to PLD1, suppressed the stimulating effect of Rho-kinase on PIP2 synthesis. These findings suggest that PLD1 is involved in the stimulation of PIP-5-kinase by Arf1 as well as by RhoA and Rho-kinase. After sucrose density gradient centrifugation of HEK-293 cell lysates, we isolated two separate PIP2 pools. PLD1 and Arf1 selectively control the non-caveolar PIP2 pool in the high density fraction, whereas PLD2 affected PIP2 in both pools. In summary, these data suggest that particularly PLD1, apparently by the production of PA, functions as a physiological regulator of PIP-5-kinase that controls the synthesis of cellular PIP2 downstream to Arf1 and RhoA.

Die Integrinrezeptor-vermittelte Zelladhäsion wird durch intrazelluläre Signalkaskaden kontrolliert. Cytohesin-1 ist ein integrinbindendes Protein und ein Guanin-Nukleotid- Austauschfaktor (GEF). Dieser aktiviert das b2-Integrin LFA-1 und induziert dessen Bindung an ICAM-1. Cytohesin-1 enthält eine PH-Domäne, diese ist an der funktionalen Regulation des Proteins beteiligt und vermittelt die Membranrekrutierung über Phosphatidylinositol- (3,4,5)-trisphosphat, dem Produkt der Phosphatidylinositol-3-Kinase. Die Phosphoinositidvermittelte Membranbindung wird primär von der PH-Domäne bewirkt, jedoch wird diese Funktion von der carboxyterminalen polybasischen c-Domäne gestützt. In der vorliegenden Studie wurde gezeigt, daß ein Serin/Threonin-Motiv innerhalb dieser c-Domäne durch gereinigte PKCd in vitro und in vivo nach Phorbolesterstimulierung phosphoryliert wird. Biochemische und funktionale Analysen zeigten, daß phosphoryliertes Cytohesin-1 mit dem Aktinzytoskelett assoziiert. Weiterhin konnte gezeigt werden, daß durch Phosphorylierung von Cytohesin-1 der Guanin-Nukleotid-Austausch an ARF1 in vitro reguliert wird. ARF-Proteine sind entscheidend an der Zytoskelettreorganisation beteiligt, die während Zelladhäsionsprozessen stattfindet. In Zellen zeigte sich, daß die LFA-1-abhängige Zelladhäsion an ICAM-1 durch phosphoryliertes Cytohesin-1 drastisch gesteigert wird. Zusammengefaßt zeigen diese Erkenntnisse, daß intrazelluläre Signalkaskaden über Phosphatidylinositol-3-Kinase und Protein-Kinase-C in Cytohesin-1 als funktionalem Integrator münden. Cytohesin-1 reguliert über diese Prozesse die b2-Integrin-vermittelte Zelladhäsion von T-Lymphozyten.