The yeast Saccharomyces cerevisiae nucleoporin Nup116p serves as a docking site for both nuclear import and export factors. However, the mechanism for assembling Nup116p into the nuclear pore complex (NPC) has not been resolved. By conducting a two-hybrid screen with the carboxy (C)-terminal Nup116p region as bait, we identified Nup82p. The predicted coiled-coil region of Nup82p was not required for Nup116p interaction, making the binding requirements distinct from those for the Nsp1p-Nup82p-Nup159p subcomplex (N. Belgareh, C. Snay-Hodge, F. Pasteau, S. Dagher, C. N. Cole, and V. Doye, Mol. Biol. Cell 9:3475-3492, 1998). Immunoprecipitation experiments using yeast cell lysates resulted in the coisolation of a Nup116p-Nup82p subcomplex. Although the absence of Nup116p had no effect on the NPC localization of Nup82p, overexpression of Cterminal Nup116p in a nup116 null mutant resulted in Nup82p mislocalization. Moreover, NPC localization of Nup116p was specifically diminished in a nup82-⌬108 mutant after growth at 37°C. Immunoelectron microscopy analysis showed Nup116p was localized on both the cytoplasmic and nuclear NPC faces. Its distribution was asymmetric with the majority at the cytoplasmic face. Taken together, these results suggest that Nup82p and Nup116p interact at the cytoplasmic NPC face, with nucleoplasmic Nup116p localization utilizing novel binding partners.Nuclear pore complexes (NPCs) are massive multiprotein structures embedded in the nuclear envelope (NE), which serve as portals for regulating the traffic of macromolecules between the cytoplasm and the nucleus (52). Three-dimensional structural information for yeast Saccharomyces cerevisiae and vertebrate NPCs has been recently revealed by a combination of high-resolution cryoelectron and scanning electron microscopy (EM) analysis (2,3,16,27,46,47,69). NPCs possess a central plug surrounded by eight spokes which attach to cytoplasmic and nuclear rings. These rings anchor the peripherally associated cytoplasm filaments and nuclear basket. Yeast S. cerevisiae NPCs are comprised of ϳ30 different proteins, termed nucleoporins (48). Models of NPC structure have predicted that the distinct modular structures of the NPC are formed from subsets of distinct nucleoporin subunits. In support of this, subcomplexes containing different nucleoporins have been biochemically isolated and characterized (for example, references 6, 9, 14, 21 to 23, 25, 30, 40, 41, and 58). Moreover, immunoelectron microscopy (IEM) experiments have localized some nucleoporins to exclusively the cytoplasmic filaments or the nuclear basket and others to either symmetric or asymmetric distributions on the central core structure (reviewed in references 48 and 60). This differential localization may reflect distinct roles for particular nucleoporins in mediating particular steps of the nuclear transport mechanism.One strategy for dissecting the hierarchy of protein-protein interactions that account for NPC structure and function has been to analyze yeast S. cerevisiae mutants. Mutations...

Polypeptide binding by the chaperone Hsp70 is regulated by its ATPase activity, which is itself regulated by co-chaperones including the Bag domain nucleotide exchange factors. Here, we tested the functional contribution of residues in the Bag domain of Bag-1M that contact Hsp70. Two point mutations, E212A and E219A, partially reduced co-chaperone activity, whereas the point mutation R237A completely abolished activity in vitro. Molecular chaperones of the heat shock protein 70-kDa (Hsp70) 1 family are conserved from Escherichia coli to mammals, and assist the folding of newly synthesized polypeptides as well as the refolding of proteins denatured under stress (1-3). All family members contain a ϳ44-kDa N-terminal ATPase domain and a ϳ25-kDa C-terminal peptide binding domain. Biochemically, the best characterized Hsp70 proteins are mammalian cytosolic Hsc70 (the constitutively expressed form) and DnaK of the E. coli cytosol. For both of these chaperones, the ATPase cycle has been shown to control the binding and release of substrate peptides by the C-terminal domain (2).Hsp70 chaperones alternate between two nucleotide-bound states; the ATP-bound form has low affinity for substrate with high on-and off-rates of peptide binding, whereas the ADPbound state has high affinity for substrate with slow on-and off-rates (4 -7). Peptide is first bound by Hsp70 in the ATP state. Hydrolysis of ATP induces conformational rearrangements within the C-terminal peptide binding domain that encloses peptide between a lidlike helical subdomain and a ␤-sheet structure that forms a binding cleft (8, 9). Exchange of ADP for a fresh ATP molecule then converts the peptide binding domain to an open state, allowing efficient release of the bound peptide (4, 5).The steady-state ATPase rate (0.02-0.2 min Ϫ1

Integral membrane proteins are predicted to play key roles in the biogenesis and function of nuclear pore complexes (NPCs). Revealing how the transport apparatus is assembled will be critical for understanding the mechanism of nucleocytoplasmic transport. We observed that expression of the carboxyl-terminal 200 amino acids of the nucleoporin Nup116p had no effect on wild-type yeast cells, but it rendered the nup116 null strain inviable at all temperatures and coincidentally resulted in the formation of nuclear membrane herniations at 23 degrees C. To identify factors related to NPC function, a genetic screen for high-copy suppressors of this lethal nup116-C phenotype was conducted. One gene (designated SNL1 for suppressor of nup116-C lethal) was identified whose expression was necessary and sufficient for rescuing growth. Snl1p has a predicted molecular mass of 18.3 kDa, a putative transmembrane domain, and limited sequence similarity to Pom152p, the only previously identified yeast NPC-associated integral membrane protein. By both indirect immunofluorescence microscopy and subcellular fractionation studies, Snl1p was localized to both the nuclear envelope and the endoplasmic reticulum. Membrane extraction and topology assays suggested that Snl1p was an integral membrane protein, with its carboxyl-terminal region exposed to the cytosol. With regard to genetic specificity, the nup116-C lethality was also suppressed by high-copy GLE2 and NIC96. Moreover, high-copy SNL1 suppressed the temperature sensitivity of gle2-1 and nic96-G3 mutant cells. The nic96-G3 allele was identified in a synthetic lethal genetic screen with a null allele of the closely related nucleoporin nup100. Gle2p physically associated with Nup116p in vitro, and the interaction required the N-terminal region of Nup116p. Therefore, genetic links between the role of Snl1p and at least three NPC-associated proteins were established. We suggest that Snl1p plays a stabilizing role in NPC structure and function.

To our knowledge these are the first documented cases of TMA associated with HES. We propose that products released from degranulated eosinophils caused endothelial injury and microvascular thrombosis. Recognition of this serious renal complication associated with blood eosinophilia should prompt early diagnosis and treatment.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are CD5+ small B-cell neoplasms (SBCNs) with overlapping features. Flow cytometric immunophenotyping is often used to help differentiate CLL from MCL, and a characteristic CLL phenotype is considered essentially diagnostic. However, previous studies have not specifically examined how well a typical MCL immunophenotype distinguishes MCL from CLL. We identified 28 cases of SBCN with typical flow cytometry-determined MCL immunophenotypes consisting mostly of peripheral blood and bone marrow specimens. Fluorescence in situ hybridization analysis indicated that 57% (16/28) had t(11;14) translocations consistent with MCL, while 32% (9/28) lacked t(11;14) translocations but harbored other cytogenetic abnormalities commonly found in CLL. There were no significant morphologic or immunophenotypic differences between the t(11;14)-positive and t(11;14)-negative cases. Our findings suggest that many blood-based SBCNs with typical MCL immunophenotypes likely represent cases of phenotypically atypical CLL, which would have important clinical implications.