The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.

Assembly of DNA parts into DNA constructs is a foundational technology in the emerging field of synthetic biology. An efficient DNA assembly method is particularly important for high-throughput, automated DNA assembly in biofabrication facilities and therefore we investigated one-step, scarless DNA assembly via ligase cycling reaction (LCR). LCR assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a thermostable ligase to join DNA backbones, and multiple denaturation-annealing-ligation temperature cycles to assemble complex DNA constructs. The efficiency of LCR assembly was improved ca. 4-fold using designed optimization experiments and response surface methodology. Under these optimized conditions, LCR enabled one-step assembly of up to 20 DNA parts and up to 20 kb DNA constructs with very few single-nucleotide polymorphisms (<1 per 25 kb) and insertions/deletions (<1 per 50 kb). Experimental comparison of various sequence-independent DNA assembly methods showed that circular polymerase extension cloning (CPEC) and Gibson isothermal assembly did not enable assembly of more than four DNA parts with more than 50% of clones being correct. Yeast homologous recombination and LCR both enabled reliable assembly of up to 12 DNA parts with 60-100% of individual clones being correct, but LCR assembly provides a much faster and easier workflow than yeast homologous recombination. LCR combines reliable assembly of many DNA parts via a cheap, rapid, and convenient workflow and thereby outperforms existing DNA assembly methods. LCR assembly is expected to become the method of choice for both manual and automated high-throughput assembly of DNA parts into DNA constructs.

We have established that two homologous nucleoporins, Nup170p and Nup157p, play an essential role in the formation of nuclear pore complexes (NPCs) in Saccharomyces cerevisiae. By regulating their synthesis, we showed that the loss of these nucleoporins triggers a decrease in NPCs caused by a halt in new NPC assembly. Preexisting NPCs are ultimately lost by dilution as cells grow, causing the inhibition of nuclear transport and the loss of viability. Significantly, the loss of Nup170p/Nup157p had distinct effects on the assembly of different architectural components of the NPC. Nucleoporins (nups) positioned on the cytoplasmic face of the NPC rapidly accumulated in cytoplasmic foci. These nup complexes could be recruited into new NPCs after reinitiation of Nup170p synthesis, and may represent a physiological intermediate. Loss of Nup170p/Nup157p also caused core and nucleoplasmically positioned nups to accumulate in NPC-like structures adjacent to the inner nuclear membrane, which suggests that these nucleoporins are required for formation of the pore membrane and the incorporation of cytoplasmic nups into forming NPCs.

In humans and rodents, the lysosomal catabolism of core Man 3 GlcNAc 2 N-glycan structures is catalyzed by the concerted action of several exoglycosidases, including a broad specificity lysosomal ␣-mannosidase (LysMan), core-specific ␣1,6-mannosidase, ␤-mannosidase, and cleavage at the reducing terminus by a di-Nacetylchitobiase. We describe here the first cloning, expression, purification, and characterization of a novel human glycosylhydrolase family 38 ␣-mannosidase with catalytic characteristics similar to those established previously for the core-specific ␣1,6-mannosidase (acidic pH optimum, inhibition by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, and stimulation by Co 2؉ and Zn 2؉). Substrate specificity studies comparing the novel human ␣-mannosidase with human LysMan revealed that the former enzyme efficiently cleaved only the ␣1-6mannose residue from Man 3 GlcNAc but not Man 3 GlcNAc 2 or other larger high mannose oligosaccharides, indicating a requirement for chitobiase action before ␣1,6-mannosidase activity. In contrast, LysMan cleaved all of the ␣-linked mannose residues from high mannose oligosaccharides except the core ␣1-6mannose residue. ␣1,6-Mannosidase transcripts were ubiquitously expressed in human tissues, and expressed sequence tag searches identified homologous sequences in murine, porcine, and canine databases. No expressed sequence tags were identified for bovine ␣1,6-mannosidase, despite the identification of two sequence homologs in the bovine genome. The lack of conservation in 5-flanking sequences for the bovine ␣1,6-mannosidase genes may lead to defective transcription similar to transcription defects in the bovine chitobiase gene. These results suggest that the chitobiase and ␣1,6-mannosidase function in tandem for mammalian lysosomal N-glycan catabolism.

Structural aspects of lipoarabinomannans (LAM) from Mycobacterium tuberculosis and Mycobacterium smegmatis were investigated by using mild acid hydrolysis in combination with Fourier-transform ion cyclotron resonance (FT-ICR), and quadrupole ion trap mass spectrometry. Exact mass measurements with less than 2.5 ppm mass error confirmed the presence of a series of arabinose oligomers (Ara n ; n ϭ 2-7) as the major components observed following mild acid hydrolysis of both M. tuberculosis and M. smegmatis LAM. However, the mass spectrum of the resulting LAM extract also revealed a highly-abundant distribution of ions that exact mass measurements identified as mannose-linked arabinose species, Ara n Man m ϩ Na ϩ (n ϭ 1-6; m ϭ 1-3). The observed mannose caps were linked to arabinose species as mono-, di-, and trimannose units, and the ratio of the mono-, di-, and trimannose caps was determined to be 1.00:9.00:1.15, respectively, different from previous reports. Analysis of the linkage of lithiated arabinose trimer standards was accomplished with MS 3 experiments and the information generated was used to identify linkages of arabinose trimers generated by mild acid hydrolysis of M. tuberculosis and M. smegmatis LAM. The MS 3 spectra confirmed the linkage of arabinose trimers from M. smegmatis and M. tuberculosis LAM as predominantly