Arf gtpase activator

At the indicated times, cells were removed and fixed. Transfected cells were detected by using a monoclonal antibody to the FLAG epitope and were counterstained with rhodamine-phalloidin. A cell was considered spread when the total diameter was 2-fold or greater than the nuclear diameter.

The data are the means and range for two experiments. Spreading was determined at 15 min. Dorsal ruffles were visualized by using rhodamine-phalloidin. Cells were transfected in parallel to those used for spreading and ruffling assay and lysed in RIPA buffer 0.

Cell spreading was studied during adhesion to fibronectin. Like the endogenous protein, the ectopically expressed ASAP1 was at the edge of the cell during cell spreading.

However, the overexpressed ASAP1 persisted at the edge for a longer period, remaining evident at 1 to 2 h Fig. In the low-power field shown in Fig. As shown in Fig. Arginine at position , highly conserved among proteins known to have Arf GAP activity 45 , was changed to lysine. In contrast, mutation of conserved cysteine to serine interfered with both PZA structure and activity.

The differences were unlikely caused by differences in levels of expression, because the fraction of cells transfected and the levels of overexpression Fig. These results implicate an Arf pathway as a regulator of cytoskeleton reorganization and a target of PDGF-mediated signaling. Given the interaction of ASAP1 with other regulators of the cytoskeleton, phosphoinositides and Src, the role of ASAP1 may be to integrate these signals, ensuring that membrane traffic and cytoskeletal changes are coordinated at each step of the cycle of events that result in cell movement.

Second, the effect of overexpressing ASAP1 suggests a regulatory role. Of these Arfs, Arf6 has been implicated in plasma membrane events, and both Arf1 and Arf6 have been implicated as regulators of the cytoskeleton. Arf proteins have been found to activate phospholipase D 46 , 47 and phosphoinositide kinases 48 — 51 and to mediate PDGF-induced phospholipase D activation In addition, Arf proteins are affected by phosphoinositides.

Both Arf exchange factors 10 , 53 — 56 and GAPs 27 , 28 , 42 contain PH domains and are activated by phosphoinositides. Thus, PtdInsP 2 could function as a second messenger with effects on the cytoskeleton mediated by actin-binding proteins or Rho family proteins A system of feedforward and feedback loops impinging on Arf would regulate PtdInsP 2 production.

ASAP1 might also affect the activity of Rho family proteins with consequent changes in the cytoskeleton. Interaction between the activities of Arf and Rho family proteins has been noted previously 16 , Because both Arf and Rho regulate phospholipase D 58 and phosphatidylinositol 4-monophosphate PtdIns4P 5 kinase 51 , the mechanisms described in the preceding paragraph might be involved. However, Arf and Rho family proteins could interact in other ways as well. For instance, Arf could affect the trafficking of Rac 16 , or Rho and Arf could together affect the activity of a common protein, such as Partner of Rac 11 , 59 , PtdIns4P 5 kinase 60 , or phospholipase D Alternatively, Arf and Rho proteins could reciprocally affect each other's activation or inactivation.

Conceivably, Arf could affect Rho family proteins in a manner similar to the effect of heterotrimeric G proteins on this family through p Conversely, the GAP activity may be regulated through the interactions. In either case, ASAP1 could function to integrate these various signals, thereby ensuring that membrane traffic and cytoskeletal changes are coordinated.

We thank Drs. Thomas Parsons, and Ken Yamada for discussions and advice; Dr. Thomas Parsons for sharing unpublished data; and Jenny Clark for expertise in nucleotide sequencing. Article published online before print: Proc. USA, Article and publication date are at www. National Center for Biotechnology Information , U. Published online Mar Paul A. Megan T. Jonathan A. Author information Article notes Copyright and License information Disclaimer. E-mail: vog. Received Dec This article has been cited by other articles in PMC.

Abstract Arf family GTP-binding proteins are best characterized as regulators of membrane traffic, but recent studies indicate an additional role in cytoskeletal organization. Materials and Methods Antibodies. Open in a separate window. Figure 1. Assays of Cytoskeletal Remodeling. Figure 2. Figure 3. Acknowledgments We thank Drs. References 1. Bretscher M S. Trends Cell Biol. Gumbinar B M.

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Kam, J. Breuer D, Wagener C. Exp Cell Res. R Golgi stack located in vesicle cluster with gold labelled vesicles; enlarged inset showing single gold-labeled vesicle. S, T Root tip region, comparable to F overview. T Enlarged boxed area of S showing weak clustering of fluorescent spots. U TEM image of representative region with clustered gold signal, located at the TGN; enlarged inset showing two gold-labelled vesicles.

V, W Root tip region, comparable to F overview. W Enlarged boxed area of S showing stronger clustering of fluorescent spots. X TEM image of representative region with gold signal in clustered vesicles; enlarged inset showing gold-labelled vesicle.

Abbreviations: c, cortex; cw, cell wall; e, epidermis; er, endoplasmic reticulum; erc, ER-connected compartment; g, Golgi stack; n, nucleus; rc, root cap; t, trans-Golgi network; v, vesicle; va, vacuole. Tubulin staining visualized phragmoplasts during cytokinesis J, N; cyan. Blue, DAPI-stained nuclei. The same wild-type controls were used as in Figs 3 and 5 and S3. Col-0, wild-type control. The tryptophan fluorescence was measured at an interval of 5 seconds, using the excitation and emission wavelength of nm and nm, respectively.

See also S10 Data. Sheet 1 contains the relevant candidates taken from the complete list of identification in sheet 2. Sheet 2 contains identified protein groups sorted by gene names and presented together with their iBAQ intensity based absolute quantification: sum of peak intensities of all peptides matching to a specific protein divided by the number of theoretically observable peptides values.

Abstract In eukaryotes, GTP-bound ARF GTPases promote intracellular membrane traffic by mediating the recruitment of coat proteins, which in turn sort cargo proteins into the forming membrane vesicles.

Author summary Membrane traffic plays an important role in cellular homeostasis, cell-cell communication, nutrient uptake and organismal interaction, delivering membrane proteins as well as secreted proteins to their sites of action and degradation. Introduction ARF GTPases and their guanine-nucleotide exchange factors ARF-GEFs play essential roles in the formation of membrane vesicles that transport cargo proteins from donor to acceptor compartments within the endomembrane system.

Download: PPT. Fig 1. Are ARF proteins of the different classes essential? Fig 2. Fig 3. Fig 4. Fig 5. Fig 6. Fig 7. Fig 8. Discussion Our results strongly suggest that, in contrast to mammals and yeast, Arabidopsis can successfully carry out essential membrane trafficking with only one class of ARF GTPase, represented by isoforms of ARF1, and that this single class of ARF GTPase can be activated by all eight ARF-GEFs acting in different trafficking pathways—secretion including cytokinesis and vacuolar traffic , endocytosis, and recycling Fig 8.

Cloning of constructs for transient expression in protoplasts Expression cassette generation: The vector pFF04 [ 47 ] was modified to contain a mas promoter sequence, NLS-GFP , and a 35S terminator sequence. Immunofluorescence localization and live imaging Five days old seedlings were incubated in 1 ml liquid growth medium 0. Immunoprecipitation Arabidopsis seedlings 5—6 days old were ground in liquid nitrogen and suspended in lysis buffer 50mM Tris pH 7.

Quantitative transport assays Protoplasts were prepared and electrotransfected as previously done [ 47 ]. Supporting information. S1 Fig. S2 Fig. S3 Fig. S4 Fig. S5 Fig. S6 Fig. Secretion of xyloglucan to the extracellular space is affected by interference with ARF1 function. S7 Fig. S8 Fig.

S9 Fig. Expression of recombinant proteins and in vitro exchange assay. S1 Table. S2 Table. S3 Table. S1 Data.

Numerical data related to Fig 2A. S2 Data. Numerical data related to Fig 2Q. S3 Data. Numerical data related to Fig 2R.

S4 Data. Numerical data related to Fig 2S. S5 Data. Numerical data related to Fig 6K. S6 Data. Numerical data related to Fig 7. S7 Data. Numerical data related to S1C Fig. S8 Data.

Numerical data related to S4J Fig. S9 Data. Numerical data related to S4L Fig. S10 Data. Numerical data related to S9 Fig. References 1. Large ARF guanine nucleotide exchange factors in membrane trafficking.

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