BIME joins the destruction team

YAMASHITA, Y. M. et al. (1996) 205 cyclosome complex formation and proteolytic

activity inhibited by the cAMP/PKA pathway Nature 384, 276-279

PETERS, J-M., KING, R. W., HOOG, C. and KIRSCHNER, M. W. (1996)

Identification of BIME as a subunit of the anaphase-promoting complex

Science 274, 1199-l 201

ZACHARIAE, W., SHIN, T. H., CALOVA, M., OBERMAIER, B. and NASMYTH, K. (1996)

Identification of subunits of the anaphase-promoting complex of Saccharomyces cerevisiae

Science 274, 1201-l 204

Destruction of the mitotic cyclins and entry into anaphase depend on a ubiquitin-protein ligase activity that is acti- vated during mitosis and is part of an -205 complex.called the cyclosome or anaphase-promoting complex (APC). APC purified from Xenopus egg extracts is composed of at least eight different subunits, two of which were immuno- identified previously as homologues of Cdcl6p and Cdc27p, proteins essential for the metaphase+anaphase transition in both yeast and mammalian cells. The APC may also con- tain a homologue of Cdc23p as Cdc23p is known to form a complex with Cdcl6p and Cdc27p in Saccharomyces cerevisiae, and CDCl6 and CDC23 were identified as genes required for proteolysis of cyclin B in yeast. These papers identify homologues of B/ME, a gene required for ana- phase in Aspergillus nidulans, as the largest subunit of the cyclosome/APC.

Peters et a/. microsequenced individual subunits of the Xenopus APC and confirmed that Cdcl6p, Cdc23p and Cdc27p are part of this complex. The largest subunit of the APC, APCl, was most similar to Tsg24 from mouse, a pro- tein similar to BIME of A. nidulans. Antibodies against Tsg24 cross-reacted with APCl, and the protein recognized by these antibodies cofractionated with Xenopus Cdcl6p and Cdc27p. Yamashita and colleagues cloned the c&4+ gene from Schizosaccharomyces pombe and found it encoded a protein similar to BIME. The cut4 protein cosedimented with cut9 and nuc2 (5. pombe homologues of Cdcl6p and Cdc27p, respectively), and antibodies to cut4 coimmuno- precipitated these proteins. cut4 was also required for mitotic cyclin ubiquitination. Zachariae et al. identified an 5. cerevisiae homologue of B/ME, called APCI, in a screen for genes required for mitotic cyclin degradation. The authors found that the mitotic cyclin Clb2p accumulated in apcl- 1 mutants and that extracts prepared from apcl- I mutant cells were defective in the ubiquitination of Clb2p and Clb3p. Apcl p was found in a complex with Cdcl6p, Cdc23p and Cdc27p, suggesting that it is part of the yeast APC. 5. cerevisiae apcl-1 mutants delayed entry into anaphase, and, in A. nidulans, mutations in bimf can bypass the interphase arrest that is induced by mutation of nimA or by the presence of unreplicated DNA. These results sug- gest that, in addition to regulating exit from mitosis and the initiation of anaphase, the cyclosome/APC may nega- tively regulate entry into mitosis.

The ins and outs of HIV endocytosis

ECAN, M. A., CARRUTH, L. M., ROWELL, J. F., YU, X. and SILICIANO, R. F. (1996)

Human immunodeficiency virus type 1 envelope protein endocytosis mediated by a highly conserved intrinsic

internalization signal in the cytoplasmic domain of gp41 is suppressed in the presence of the Pr55W precursor protein

1. Viral. 70, 6547-6556

The envelope glycoprotein (Env) of human immuno- deficiency virus type 1 (HIV-l) presents something of a paradox. Its presence on the cell surface at high levels is necessary for efficient production of infectious virions, yet it is known to contain two conserved internalization motifs in its cytoplasmic tail. These YXX0 motifs are also found in proteins such as the transferrin receptor (Tf-R) and are known to be used by the Env protein, which is rapidly endocytosed when expressed on the surface of cells.

The authors use fluorescence-activated cell sorting (FACS) analysis to monitor the presence of Env and Tf-R on the cell surface and show in CD4+ cell lines that Env is endocytosed, albeit less efficiently than is the Tf-R. However, in HIV-l- infected CD4+ lymphoblasts, Env remains on the cell surface, implying that a viral factor regulates endocytosis. This viral factor turns out to be the MA domain of the Gag protein. When coexpressed with Env, Gag prevents internalization of Env but has no effect on Tf-R. For the virus, Env expression on the cell surface has several undesirable outcomes, includ- ing Env-dependent syncytium formation and other toxic events. Thus the internalization serves to limit these toxic effects. When the time comes for virus assembly and bud- ding, the Gag protein then comes into the picture. In theory, Gag interferes with internalization, resulting in a high concentration of Env on the cell surface and an efficient incorporation into virions. Just how this interference with endocytosis occurs is not known. What is intriguing, however, is the idea that it somehow comes between the tyrosine- containing internalization sequence and the P2 chain of the AP-2 adaptor proteins, which are the mediators of clathrin- dependent protein sorting in the endocytic pathway.

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