Cut and paste

HEADLINES

New way out of yeast CLEVES, A. E., COOPER, D. N. W., BARONDES, S. H. and KELLY, R. B. (1996) A new pathway for protein export in Saccharomyces cerevisiae j. Cell Biol. 133, 1017-I 026 Mammalian cells exhibit at least two different pathways for secretion. The major (classical) pathway starts with the translocation of proteins across the endoplasmic reticulum (ER) membrane, followed by vesicular transport to and through the Colgi stacks and’trans Colgi network (TGN), and eventually the vesicle-mediated release of cargo at the plasma membrane into the extracellular environment. Little is known about the nonclassical pathway, but recent investigations revealed that the secretion of interleukin 1 j3, thioredoxin, basic fibroblast growth factor (bFCF) and galectin-1 is not affected by inhibitors for the classical pathway such as monensin or brefeldin A. Cleves et al. were interested in whether a similar nonclassical export pathway also exists in the yeast Saccharomyces cerevisiae. In a set/P strain under conditions where the classical pathway is blocked, a number of endogenous proteins are still secreted. To find out more, the authors expressed the mammalian nonclassical export substrate galectin-1, a 14-kDa lactose-binding lectin, in yeast. Their data indicate that galectin-1 is secreted and remains attached to the extracellular plasma membrane, from were it can be extracted and quantitated. The secretion was found to be independent of Ste6p, which transports the 12-amino-acid lipopeptide a factor from the cytosol to the periplasm. This assay was then used to screen for gene products involved in nonclassical transport. Two novel gene products were found. NCEI seems to be a nonclassical export substrate, whereas NCU is a potential transporter. This work shows nicely that there is an alternative secretory pathway in yeast. Identification of novel players in yeast and, eventually, in mammalian systems will lead hopefully to a better understanding of this poorly understood ‘nonclassical’ event.

central cell. Signalling between R8 and the surrounding cells is necessary for correct eye development. R8 expresses the ligand for the SEVENLESS tyrosine kinase receptor, namely BRIDE OF SEVENLESS, or BOSS. It has been shown previously that R7 can endocytose BOSS from the R8 &asma membrane in a process that removes activated ligand-receptor complexes from the R7 plasma membrane. BOSS is then delivered to an endocytic compartment similar to a multivesicular body (MVB). In this report, the authors identify a gene required for this step in BOSSSEVENLESS downregulation - Hook. hook mutant flies exhibit pleiotropic defects including abnormal (hooked) bristle morphology and eye degeneration. Endocytosis of BOSS from R8 to R7 cells can be observed by staining Drosophila eye imaginal discs with antibodies against BOSS. In normal flies, the BOSS staining pattern shows strong staining of R8 cells and also staining of an intracellular structure in R7 cells. In hook flies, only BOSS staining in R8 cells is visible. Analysis of the Hook gene indicates that it encodes a 679amino-acid protein with a large, predicted coiled-coil domain. Expression of epitope-tagged truncated forms of the protein showed that HOOK forms a homodimer and that the coiled-coil domain is responsible for this dimerization. The requirement for HOOK in R7 cells for the endocytosis of BOSS was demonstrated by placing the expression of Hook under the control of a sev enhancer cassette, which directed expression in R7 cells but not R8. Crossing these flies back to hookflies restored endocytosis of BOSS into R7 cells. HOOK was localized to large vacuolar structures that were also positive for endocytosed fluorescent dextran and BOSS. The exact role of HOOK in endocytosis remains to be established and may require an in vitro approach. As HOOK is not required for viability, it seems likely that HOOK may act downstream of internalization, possibly in the sorting of BOSS into MVBs.

rCut and paste

#e <

Hooking the BOSS into endocytosis KRAMER, H. and PHISTRY, M. (1996) Mutations in the Drosophila hook gene inhibit endocytosis of the Boss transmembrane ligand into multivesicular bodies 1. Cell Biol. 133, 1205-1215 The compound eye of Drosophila provides a useful model for studying the importance of cell-cell interactions in development. Photoreceptor cells (R-cells) of individual ommatidia are arranged in a characteristic pattern consisting of seven cells (RI-R7) surrounding and in contactwith R8, the trends in CELL BIOLOGY (Vol. 6) September 1996

FUNABIKI, H., YAMANO, H., KUMADA, K., NAGAO, K., HUNT, T. and YANACIDA, M. (1996) Cut2 proteolysis required for sister-chromatid separation in fission yeast Nature 381, 438-441 The avalanche of work that implicated regulated cyclin B accumulation and destruction in the entry into and exit out of mitosis made it tempting to suppose that cyclin abundance was all that mattered in mitosis. Cyclin B is destroyed by ubiquitination-dependent proteolysis at the metaphaseanaphase transition, but preventing the destruction of cyclin B leads to an arrest after anaphase. Further experiments suggested that the ubiquitination-dependent proteolysis of other proteins is the critical event that leads to sisterchromatid separation. The cut temperature-sensitive mutants were identified in fission yeast based on the observation of guillotined nuclei at the restrictive temperature, which arise as a result of septation in the absence of chromosome segregation. Funabiki et al. determine that Cut2 protein is located in the nucleus

This month’s headlineswere contributed by lsabelleDelumeau, Paul Ferrigno, David Hatton, Diane Hatton, Frank Peter, Matt Seaman, SylvieTourner and Bill Wells.

333

w

HEADLINES

in interphase, localized to the spindle in early to mid-mitosis and is destroyed around the time of anaphase onset. As is true for cyclin B, Cut2 is stable in C2-arrested cells, but unstable in Cl -arrested cells. Cyclin destruction in budding yeast is dependent on Cdcl6, a component of the cyclosome/anaphase-promoting complex (APC), and the instability of Cut2 in Gl is dependent on Cut9, the fission yeast homologue of Cdcl6. The destruction also requires two destruction boxes at the N-terminus of Cut2, and a truncated version of Cut2 accumulates and prevents sister-chromatid separation while the rest of the cell cycle progresses. Is Cut2 the sought-after sister-chromatid glue? If this were the case, a null mu ant should show premature sisterchromatid separation, but these mutants instead fail to separate sister chromatids, just like the cells that express the truncated, indestructible Cut2. Funabiki et al. have also observed that Cut2 is in a complex with Cutl, and, based on this, they propose that Cut2 must fulfil two functions: after first activating Cut1 so that Cut1 can later promote sisterchromatid separation, it must then be destroyed so that Cut1 can express that activity. This model fits the data available, but far more evidence is needed before we can tell whether the model is correct or how close Cut2 is to the action in gluing chromosomes together.

in the retina. Other features of these mice were female sterility probably due to a maturation arrest of ovarian follicles, and, as in the Rb-deficient mice, a predisposition to develop pituitary tumours. Interestingly, Nakayama et al. demonstrate that cell-cycle arrest in response to TGF-j3, rapamycin or contact inhibition is unaltered in p27Ki@-deficient mice, suggesting that ~27~‘P’ is not required in these pathways. In summary, these findings indicate that ~27~@’ is an important growth regulator for a variety of cell types.

f?

Dealing

with

sticky

patches

FRYDMAN, J. and HARTL, F. U. (1996) Principles of chaperone-assisted protein folding: differences between in vitro and in vivo mechanisms Science 272, 1497-l 502

Structural

ZHU, X. et al. (1996) analysis of substrate binding molecular chaperone DnaK Science 272, 1606-l 614

by the

r

Big mouse for p27Kip7 knockout FERO, L. et al. (1996) A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27Q”-deficient mice Cell 85, 73 3-744 NAKAYAMA, K. et al. (1996) Mice lacking ~27~@ display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors Cell 85, 707-720

Enhanced

KIYOKAWA, H. et al. (1996) growth of mice lacking the cyclin-dependent kinase inhibitor function of p27@” Cell 85, 721-732

p27’@ ’ belongs to the Cip/Kip family of cyclin-dependent kinase inhibitors that control cell-cycle progression by binding to cyclin-dependent kinase (CDK) complexes. ~27~@’ mediates Cl -phase arrest induced by transforming growth factor B (TCF-B), contact inhibition or serum deprivation in epithelial cell lines. Various lines of evidence suggested that p27@” may play a central role in the negative regulation of cell growth in a broad range of tissues. In order to explore the biological importance of ~27~‘~’ in mouse development, three laboratories have now described the phenotype of p27Q”-deficient animals. All observed that disruption of the gene encoding ~27~‘P’ resulted in mice that were bigger than their littermates. Consistent with the high level of expression of p27KiP1 in these tissues, the major abnormalities that occurred in p27Q1-deficient animals were enlargement of thymus, testis, ovary and adrenal medulla as well as a possible overgrowth of the neuroepithelium

334

Studies of protein folding have traditionally used artificially denatured model substrates, renaturation being facilitated by interactions with molecular chaperones. In this way, the paradigmatic bacterial protein chaperone DnaK (Hsp70 in eukaryotes) has been found to aid refolding of denatured substrates in an ATP-dependent manner. Zhu et al. now report the crystal structure of the polypeptide-binding domain of DnaK with a model peptide bound. They reveal an extraordinary three-dimensional structure, somewhat reminiscent of a seven-fingered hand, clutching the peptide. Consistent with predictions from peptide-binding studies, the substrate-binding cleft is tailored to recognize aliphatic residues, lending support to the idea that the role of these chaperones is to recognize hydrophobic domains that are normally buried within folded proteins or that mediate protein-protein interactions in multimeric complexes. Although captured closed, it is easy to visualize the hand opening to hold hydrophobic patches at the surface of large and/or folded polypeptides, and the observed flexibility in the hinge region may play a role in the ATP-dependent substrate-binding-and-release cycle. At least two interacting factors are known to recognize unfolded polypeptides, one comprising DnaK/Hsp70 and its cofactor DnaJ/Hsp40, and the other an oligomeric complex - GroEL in Escherichia co/i, TRiC in eukaryotes. Frydman and Hart1 present a series of elegant experiments suggesting that these two complexes may perform different roles in vivo. Their most exciting finding is that nascent polypeptides interact primarily with DnaK-DnaJ, whereas longer unfolded/denatured polypeptides can interact with GroEL/ TRiC. Release of the mature, folded polypeptide from the latter may simply result from the masking of the hydrophobic surfaces within the globular protein. Where hydrophobic surfaces need to remain exposed prior to the assembly of multi-polypeptide complexes, Hsp70-Hsp40 chaperones may play a second role, protecting these patches on the surface of folded proteins until a matching hydrophobic area is contacted. A major challenge will be to work out how this is achieved. trends in CELL BIOLOGY (Vol. 6) September 1996