Adp Ribosylation Factor-like Protein 2 (Arl2) Regulates the Interaction of Tubulin-Folding Cofactor D with Native Tubulin

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the α/β- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a β-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.

[1]  D Botstein,et al.  Yeast mutants sensitive to antimicrotubule drugs define three genes that affect microtubule function. , 1990, Genetics.

[2]  P. Silver,et al.  Interactions between a Nuclear Transporter and a Subset of Nuclear Pore Complex Proteins Depend on Ran GTPase , 1999, Molecular and Cellular Biology.

[3]  N. Cowan,et al.  A cytoplasmic chaperonin that catalyzes beta-actin folding. , 1992, Cell.

[4]  W. Kabsch,et al.  Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation , 1989, Nature.

[5]  Nicholas J. Cowan,et al.  Tubulin Folding Cofactors as GTPase-activating Proteins , 1999, The Journal of Biological Chemistry.

[6]  T. Toda,et al.  Functional dissection and hierarchy of tubulin-folding cofactor homologues in fission yeast. , 1999, Molecular biology of the cell.

[7]  B. Roberts,et al.  Saccharomyces cerevisiae PAC2 functions with CIN1, 2 and 4 in a pathway leading to normal microtubule stability. , 1997, Genetics.

[8]  J Vandekerckhove,et al.  A novel cochaperonin that modulates the ATPase activity of cytoplasmic chaperonin , 1994, The Journal of cell biology.

[9]  J Vandekerckhove,et al.  Pathway leading to correctly folded beta-tubulin. , 1996, Cell.

[10]  S. Lewis,et al.  The alpha- and beta-tubulin folding pathways. , 1997, Trends in cell biology.

[11]  J. Vandekerckhove,et al.  Prefoldin, a Chaperone that Delivers Unfolded Proteins to Cytosolic Chaperonin , 1998, Cell.

[12]  J. Moss,et al.  Structure and Function of ARF Proteins: Activators of Cholera Toxin and Critical Components of Intracellular Vesicular Transport Processes (*) , 1995, The Journal of Biological Chemistry.

[13]  Christophe Ampe,et al.  Pathway Leading to Correctly Folded β-Tubulin , 1996, Cell.

[14]  W. Welch,et al.  Prefoldin–Nascent Chain Complexes in the Folding of Cytoskeletal Proteins , 1999, The Journal of cell biology.

[15]  T. Toda,et al.  Essential role of tubulin‐folding cofactor D in microtubule assembly and its association with microtubules in fission yeast , 1998, The EMBO journal.

[16]  Nicholas J. Cowan,et al.  The α- and β-tubulin folding pathways , 1997 .

[17]  Frank McCormick,et al.  The GTPase superfamily: a conserved switch for diverse cell functions , 1990, Nature.

[18]  E. Nogales,et al.  Alf1p, a Clip-170 Domain-containing Protein, Is Functionally and Physically Associated with ␣ -tubulin , 1999 .

[19]  M. Kirschner,et al.  Beyond self-assembly: From microtubules to morphogenesis , 1986, Cell.

[20]  R. Klausner,et al.  ARF: a key regulatory switch in membrane traffic and organelle structure. , 1994, Current opinion in cell biology.

[21]  F. Solomon,et al.  Rbl2p, a yeast protein that binds to β-tubulin and participates in microtubule function in vivo , 1995, Cell.

[22]  J. Goldberg,et al.  Structural Basis for Activation of ARF GTPase Mechanisms of Guanine Nucleotide Exchange and GTP–Myristoyl Switching , 1998, Cell.

[23]  R. Kahn,et al.  Arf proteins: the membrane traffic police? , 1995, Trends in biochemical sciences.

[24]  N. Sonenberg,et al.  The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins , 1995, Molecular and cellular biology.

[25]  D. Ringe,et al.  Structure of the human ADP-ribosylation factor 1 complexed with GDP , 1994, Nature.

[26]  W. Tap,et al.  Quasi-native Chaperonin-bound Intermediates in Facilitated Protein Folding (*) , 1995, The Journal of Biological Chemistry.

[27]  Mark S. Boguski,et al.  Proteins regulating Ras and its relatives , 1993, Nature.

[28]  F. Hartl Molecular chaperones in cellular protein folding , 1996, Nature.

[29]  R. Kahn,et al.  The ARF-like 2 (ARL2)-binding Protein, BART , 1999, The Journal of Biological Chemistry.

[30]  S. Lewis,et al.  A chaperone with a hydrophilic surface , 1999, Nature Structural Biology.

[31]  J. Battey,et al.  Selective amplification of additional members of the ADP-ribosylation factor (ARF) family: cloning of additional human and Drosophila ARF-like genes. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[32]  John O. Thomas,et al.  A cytoplasmic chaperonin that catalyzes β-actin folding , 1992, Cell.

[33]  Nicholas J. Cowan,et al.  Tubulin Subunits Exist in an Activated Conformational State Generated and Maintained by Protein Cofactors , 1997, The Journal of cell biology.

[34]  J R McIntosh,et al.  Sto1p, a fission yeast protein similar to tubulin folding cofactor E, plays an essential role in mitotic microtubule assembly. , 1999, Journal of cell science.

[35]  W. Balch,et al.  GTPases: multifunctional molecular switches regulating vesicular traffic. , 1994, Annual review of biochemistry.