The Putative “Switch 2” Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein

Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the α2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative α2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the α3 and α4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B α2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B α2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the α2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.

Replication Factor C3 of Schizosaccharomyces pombe, a Small Subunit of Replication Factor C Complex, Plays a Role in Both Replication and Damage Checkpoints

We report here the isolation and functional analysis of the rfc3+ gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3+ gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3+ is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.

The Schizosaccharomyces pombe spo20+ Gene Encoding a Homologue of Saccharomyces cerevisiae Sec14 Plays an Important Role in Forespore Membrane Formation

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20+ is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20+ gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

Plays and poetry : manuscript, [17--]

Plays and poems, chiefly satires, in support of American independence and patriotic causes.

Index to children's plays,

Books about plays and play production, puppets, costuming, folk dances and singing games: p. 1-9.

Plays for children : an annotated index.

Mode of access: Internet.

Plays.

Each play has also special t.p.

The dispensation, and other plays,

The dispensation.--The star of Bethlehem.--"Through Christmas bells."--The awakening of Barbizon.

The English and American stage.

Princeton (THX) has imperfect copy: t.p. of v. 11 wanting. Typed t.p. supplied.

Subject index to children's plays /

Mode of access: Internet.

The works of William Shakespeare : the plays edited from the folio of MDCXXIII, with various readings from all the editions and all the commentators, notes, introductory remarks, a historical sketch of the text, an account of the rise and progress of the English drama, a memoir of the poet, and an essay upon his genius /

Publisher's advertisement (12 p.) at beginning of v. 2.

The Atlantic book of modern plays /

The Philosopher of Butterbiggens / Harold Chapin -- Spreading the news / Lady Gregory -- The Beggar and the King / Winthrop Parkhurst -- Tides / George Middleton -- Ile / Eugene O'neill -- Campbell of Kilmhor / J.A. Ferguson -- The sun / John Galsworthy -- The knave Hearts / Louise Saunders -- Fame and the poet / Lord Dunsany -- The captain of the gate / Beulah Marie Dix -- Gettysburg / Percy Mackaye -- Lonesome-Like / Harold Brighouse -- Riders of the sea / John Millington Synge -- The Land of Heart's Desire / William Butler Yeats -- The Riding to Lithend / Gordon Bottomley -- Night Watches / Allan Monkhouse -- Glory of the Morning / William Ellery Leonard -- Trifles / Susan Glaspell.

Stories by American authors.

v. 6. The village convict, by C. H. White. The Denver express, by A. A. Hayes. The misfortunes of Bro' Thomas Wheatley, by Lina R. Fairfax. The heartbreak cameo, by L. W. Champney. Miss Eunice's glove, by A. Webster. Brother Sebastian's friendship, by H. Frederic.--v. 7. The bishop's vagabond, by Octave Thanet. Lost, by E. Bellamy. Kirby's coals of fire, by Louise Stockton. Passages from the journal of a social wreck, by Margaret Floyd. Stella Grayland, by J. T. McKay. The image of San Donato, by Virginia W. Johnson.--v. 8. The brigade commander, by J. W. De Forest. Split zephyr, by H. A. Beers. Zerviah Hope, by Elizabeth S. Phelps. The life-magnet, by A. A. Adee. Osgood's predicament, by Elizabeth D. B. Stoddard.--v. 9. Marse Chan, by T. N. Page. Mr. Bixby's Christmas visitor, by C. S. Gage. Eli, by C. H. White. Young Strong of "The Clarion," by Milicent W. Shinn. How old Wiggins wore ship, by Captain R. T. Coffin. "--mas has come," by L. Kip.--v. 10. Pancha, by T. A. Janvier. The ablest man in the world, by E. P. Mitchell. Young Moll's peevy, by C. A. Stephens. Manmat'ha, by C. De Kay. A daring fiction, by H. H. Boyesen. The story of two lives, by Julia Schayer.

Viewpoints in modern drama, an arrangement of plays according to their essential interest,

Annotated, with author and title index.

False premises; five one act plays,

The Christmas tree.--The torch of time: a study in revolution.--Moonshine.--A fool and his money: a wayward comedy.--The house-fairy.