Ytm1, Nop7, and Erb1 form a complex necessary for maturation of yeast 66S preribosomes

The essential, conserved yeast nucleolar protein Ytm1 is one of 17 proteins in ribosome assembly intermediates that contain WD40 protein-protein interaction motifs. Such proteins may play key roles in organizing other molecules necessary for ribosome biogenesis. Ytm1 is present in four consecutive 66S preribosomes containing 27SA2, 27SA3, 27SB, and 25.5S plus 7S pre-rRNAs plus ribosome assembly factors and ribosomal proteins. Ytm1 binds directly to Erb1 and is present in a heterotrimeric subcomplex together with Erb1 and Nop7, both within preribosomes and independently of preribosomes. However, Nop7 and Erb1 assemble into preribosomes prior to Ytm1. Mutations in the WD40 motifs of Ytm1 disrupt binding to Erb1, destabilize the heterotrimer, and delay pre-rRNA processing and nuclear export of preribosomes. Nevertheless, 66S preribosomes lacking Ytm1 remain otherwise intact.     

Nop53p, an essential nucleolar protein that interacts with Nop17p and Nip7p, is required for pre-rRNA processing in Saccharomyces cerevisiae

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form large and intriguingly organized complexes. A novel nucleolar protein, Nop53p, was isolated by using Nop17p as bait in the yeast two-hybrid system. Nop53p also interacts with a second nucleolar protein, Nip7p. A carbon source-conditional strain with the NOP53 coding sequence under the control of the GAL1 promoter did not grow in glucose-containing medium, showing the phenotype of an essential gene. Under nonpermissive conditions, the conditional mutant strain showed rRNA biosynthesis defects, leading to an accumulation of the 27S and 7S pre-rRNAs and depletion of the mature 25S and 5.8S mature rRNAs. Nop53p did not interact with any of the exosome subunits in the yeast two-hybrid system, but its depletion affects the exosome function. In pull-down assays, protein A-tagged Nop53p coprecipitated the 27S and 7S pre-rRNAs, and His-Nop53p also bound directly 5.8S rRNA in vitro, which is consistent with a role for Nop53p in pre-rRNA processing.     

Nucleolar KKE/D repeat proteins Nop56p and Nop58p interact with Nop1p and are required for ribosome biogenesis.

Different point mutations in the nucleolar protein fibrillarin (Nop1p in Saccharomyces cerevisiae) can inhibit different steps in ribosome synthesis. A screen for mutations that are synthetically lethal (sl) with the nop1-5 allele, which inhibits pre-rRNA processing, identified NOP56. An independent sl mutation screen with nop1-3, which inhibits pre-rRNA methylation, identified a mutation in NOP58. Strikingly, Nop56p and Nop58p are highly homologous (45% identity). Both proteins were found to be essential and localized to the nucleolus. A temperature-sensitive lethal mutant allele, nop56-2, inhibited many steps in pre-rRNA processing, particularly on the pathway of 25S/5.8S rRNA synthesis, and led to defects in 60S subunit assembly. Epitope-tagged constructs show that both Nop56p and Nop58p are associated with Noplp in complexes, Nop56p and Nop1p exhibiting a stoichiometric association. These physical interactions presumably underlie the observed sl phenotypes. Well-conserved homologs are present in a range of organisms, including humans (52% identity between human hNop56p and yeast Nop56p), suggesting that these complexes have been conserved in evolution.     

Nop53p is required for late 60S ribosome subunit maturation and nuclear export in yeast

We report that Ypl146cp/Nop53p is associated with pre-60S ribosomal complexes and localized to the nucleolus and nucleoplasm. In cells depleted of Nop53p synthesis of the rRNA components of the 60S ribosomal subunit is severely inhibited, with strikingly strong accumulation of the 7S pre-rRNA and a 5' extended form of the 25S rRNA. In cells depleted of Nop53p pre-60S subunits accumulate in the nucleus. However, a heterokaryon assay demonstrated that Nop53p is not transferred between nuclei, indicating that it is not released into the cytoplasm. We conclude that Nop53p is a late-acting factor in the nuclear maturation of 60S ribosomal subunits, which is required for normal acquisition of export competence. The strong accumulation of preribosomes in the Nop53p-depleted strain further suggests that it may participate in targeting aberrant preribosomes to surveillance and degradation pathways.     

Nop2p is required for pre-rRNA processing and 60S ribosome subunit synthesis in yeast.

To investigate the function of the nucleolar protein Nop2p in Saccharomyces cerevisiae, we constructed a strain in which NOP2 is under the control of a repressible promoter. Repression of NOP2 expression lengthens the doubling time of this strain about fivefold and reduces steady-state levels of 60S ribosomal subunits, 80S ribosomes, and polysomes. Levels of 40S subunits increase as the free pool of 60S subunits is reduced. Nop2p depletion impairs processing of the 35S pre-rRNA and inhibits processing of 27S pre-rRNA, which results in lower steady-state levels of 25S rRNA and 5.8S rRNA. Processing of 20S pre-rRNA to 18S rRNA is not significantly affected. Processing at sites A2, A3, B1L, and B1S and the generation of 5' termini of different pre-rRNA intermediates appear to be normal after Nop2p depletion. Sequence comparisons suggest that Nop2p may function as a methyltransferase. 2'-O-ribose methylation of the conserved site UmGm psi UC2922 is known to take place during processing of 27S pre-rRNA. Although Nop2p depletion lengthens the half-life of 27S pre-RNA, methylation of UmGm psi UC2922 in 27S pre-rRNA is low during Nop2p depletion. However, methylation of UmGm psi UC2922 in mature 25S rRNA appears normal. These findings provide evidence for a close interconnection between methylation at this conserved site and the processing step that yields the 25S rRNA.     

Rlp7p is associated with 60S preribosomes,restricted to the granular component of the nucleolus,and required for pre-rRNA processing

Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear. Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct. The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p. The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs. Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued. Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization. Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC). All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus.     

ATP10, a yeast nuclear gene required for the assembly of the mitochondrial F1-F0 complex

A yeast nuclear gene (ATP10) is reported whose product is essential for the assembly of a functional mitochondrial ATPase complex. Mutations in ATP10 induce a loss of rutamycin sensitivity in the mitochondrial ATPase but do not affect respiratory enzymes. This phenotype has been correlated with a defect in the F0 sector of the ATPase. The wild type ATP10 gene has been cloned by transformation of an atp 10 mutant with a yeast genomic library. The gene codes for a protein of Mr = 30,293. The primary structure of the ATP10 product is not related to any known subunit of the yeast or mammalian mitochondrial ATPase complexes. To further clarify the role of this new protein in the assembly of the ATPase, an antibody was prepared against a hybrid protein expressed from a trpE/ATP 10 fusion gene. The antibody recognizes a 30-kDa protein present in wild type mitochondria. The protein is associated with the mitochondrial membrane but does not co-fractionate either with F1 or with the rutamycin-sensitive F1-F0 complex. These data suggest that the ATP10 product is not a subunit of the ATPase complex but rather is required for the assembly of the F0 sector of the complex.     

Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly

In the eukaryotic secretory and endocytic pathways, transport vesicles shuttle cargo among intracellular organelles and to and from the plasma membrane. Cargo delivery entails fusion of the transport vesicle with its target, a process thought to be mediated by membrane bridging SNARE protein complexes. Temporal and spatial control of intracellular trafficking depends in part on regulating the assembly of these complexes. In vitro, SNARE assembly is inhibited by the closed conformation adopted by the syntaxin family of SNAREs. To visualize this closed conformation directly, the X-ray crystal structure of a yeast syntaxin, Sso1p, has been determined and refined to 2.1 A resolution. Mutants designed to destabilize the closed conformation exhibit accelerated rates of SNARE assembly. Our results provide insight into the mechanism of SNARE assembly and its intramolecular and intermolecular regulation.     

Characterization of ATP12, a yeast nuclear gene required for the assembly of the mitochondrial F1-ATPase.

Mitochondrial F1-ATPase is an oligomeric enzyme composed of five distinct subunit polypeptides. The alpha and beta subunits make up the bulk of protein mass of F1. In Saccharomyces cerevisiae both subunits are synthesized as precursors with amino-terminal targeting signals that are removed upon translocation of the proteins to the matrix compartment. Recently, two different complementation groups (G13, G57), consisting of yeast nuclear mutants with defective F1, have been described. Biochemical analyses indicate that the mutational block in both groups of mutants affects a critical step needed for the assembly of the alpha and beta subunits into the F1 oligomer after their transport into mitochondria. In this study the ATP12 gene representative of the nuclear respiratory-deficient mutant of S. cerevisiae (pet) complementation group G57 has been cloned and the encoded product partially characterized. The ATP12 reading frame is 975 base pairs long and codes for a protein of Mr = 36,587. The ATP12 protein is not homologous to the subunits of F1 whose sequences are known, nor does it exhibit significant primary structure similarity to any known protein. In vitro import assays indicate that ATP12 protein is synthesized as a precursor approximately 3 kDa larger than the mature protein. The mitochondrial localization of the protein has been confirmed by Western blot analysis of mitochondrial proteins with an antibody against a hybrid protein expressed from a trpE-ATP12 fusion. Fractionation of mitochondria indicates further that the ATP12 protein is either a minor component of the matrix compartment or is weakly bound to the matrix side of the inner membrane. The molecular weight of the native protein, estimated from its sedimentation properties in sucrose gradients, is at least two times larger than the monomer. This suggests that the ATP12 protein is probably part of a larger complex.     

Gamma-tubulin is required for proper recruitment and assembly of Kar9-Bim1 complexes in budding yeast

Microtubule plus-end-interacting proteins (+TIPs) promote the dynamic interactions between the plus ends (+ends) of astral microtubules and cortical actin that are required for preanaphase spindle positioning. Paradoxically, +TIPs such as the EB1 orthologue Bim1 and Kar9 also associate with spindle pole bodies (SPBs), the centrosome equivalent in budding yeast. Here, we show that deletion of four C-terminal residues of the budding yeast gamma-tubulin Tub4 (tub4-delta dsyl) perturbs Bim1 and Kar9 localization to SPBs and Kar9-dependent spindle positioning. Surprisingly, we find Kar9 localizes to microtubule +ends in tub4-delta dsyl cells, but these microtubules fail to position the spindle when targeted to the bud. Using cofluorescence and coaffinity purification, we show Kar9 complexes in tub4-delta dsyl cells contain reduced levels of Bim1. Astral microtubule dynamics is suppressed in tub4-delta dsyl cells, but it are restored by deletion of Kar9. Moreover, Myo2- and F-actin-dependent dwelling of Kar9 in the bud is observed in tub4-delta dsyl cells, suggesting defective Kar9 complexes tether microtubule +ends to the cortex. Overproduction of Bim1, but not Kar9, restores Kar9-dependent spindle positioning in the tub4-delta dsyl mutant, reduces cortical dwelling, and promotes Bim1-Kar9 interactions. We propose that SPBs, via the tail of Tub4, promote the assembly of functional +TIP complexes before their deployment to microtubule +ends.     

DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.

To identify Saccharomyces cerevisiae mutants defective in assembly or function of ribosomes, a collection of cold-sensitive strains generated by treatment with ethyl methanesulfonate was screened by sucrose gradient analysis for altered ratios of free 40S to 60S ribosomal subunits or qualitative changes in polyribosome profiles. Mutations defining seven complementation groups deficient in ribosomal subunits, drs1 to drs7, were identified. We have previously shown that DRS1 encodes a putative ATP-dependent RNA helicase necessary for assembly of 60S ribosomal subunits (T. L. Ripmaster, G. P. Vaughn, and J. L. Woolford, Jr., Proc. Natl. Acad. Sci. USA 89:11131-11135, 1992). Strains bearing the drs2 mutation process the 20S precursor of the mature 18S rRNA slowly and are deficient in 40S ribosomal subunits. Cloning and sequencing of the DRS2 gene revealed that it encodes a protein similar to membrane-spanning Ca2+ ATPases. The predicted amino acid sequence encoded by DRS2 contains seven transmembrane domains, a phosphate-binding loop found in ATP- or GTP-binding proteins, and a seven-amino-acid sequence detected in all classes of P-type ATPases. The cold-sensitive phenotype of drs2 is suppressed by extra copies of the TEF3 gene, which encodes a yeast homolog of eukaryotic translation elongation factor EF-1 gamma. Identification of gene products affecting ribosome assembly and function among the DNAs complementing the drs mutations validates the feasibility of this approach.     

Interactions between PE2, E1, and 6K required for assembly of alphaviruses studied with chimeric viruses.

During the assembly of alphaviruses, a preassembled nucleocapsid buds through the cell plasma membrane to acquire an envelope containing two virally encoded glycoproteins, E2 and E1. Using two chimeric viruses, we have studied interactions between E1, E2, and a viral peptide called 6K, which are required for budding. A chimeric Sindbis virus (SIN) in which the 6K gene had been replaced with that from Ross River virus (RR) produced wild-type levels of nucleocapsids and abundant PE2/E1 heterodimers that were processed and transported to the cell surface. However, only about 10% as much chimeric virus as wild-type virus was assembled, demonstrating that there is a sequence-specific interaction between 6K and the glycoproteins required for efficient virus assembly. In addition, the conformation of E1 in the E2/E1 heterodimer on the cell surface was different for the chimeric virus from that for the wild type, suggesting that one function of 6K is to promote proper folding of E1 in the heterodimer. A second chimeric SIN, in which both the 6K and E1 genes, as well as the 3' nontranslated region, were replaced with the corresponding regions of RR also resulted in the production of large numbers of intracellular nucleocapsids and of PE2/E1 heterodimers that were cleaved and transported to the cell surface. Budding of this chimera was severely impaired, however, and the yield of the chimera was only approximately 10(-7) of the SIN yield in a parallel infection. The conformation of the SIN E2/RR E1 heterodimer on the cell surface was different from that of the SIN E2/SIN E1 heterodimer, and no interaction between viral glycoproteins and nucleocapsids at the cell plasma membrane could be detected in the electron microscope. We suggest that proper folding of the E2/E1 heterodimer must occur before the E2 tail is positioned properly in the cytoplasm for budding and before heterodimer trimerization can occur to drive virus budding.     

Some assembly required: yeast septins provide the instruction manual

Septins are a family of conserved proteins that form hetero-oligomeric complexes that assemble into filaments. The filaments can be organized into linear arrays, coils, rings and gauzes. They serve as membrane-associated scaffolds and as barriers to demarcate local compartments, especially for the establishment of the septation site for cytokinesis. Studies in budding and fission yeast have revealed many of the protein-protein interactions that govern the formation of multi-septin complexes. GTP binding and phosphorylation direct the polymerization of filaments that is required for septin-collar assembly in budding yeast, whereas a homolog of anillin instructs timely formation of the ring of septin filaments at the medial cortex in fission yeast. These insights should aid understanding of the organization and function of the diverse septin structures in animal cells.     

Spb1p is a yeast nucleolar protein associated with Nop1p and Nop58p that is able to bind S-adenosyl-L-methionine in vitro

We present here the characterization of SPB1, an essential yeast gene that is required for ribosome synthesis. A cold-sensitive allele for that gene (referred to here as spb1-1) had been previously isolated as a suppressor of a mutation affecting the poly(A)-binding protein gene (PAB1) and a thermosensitive allele (referred to here as spb1-2) was isolated in a search for essential genes required for gene silencing in Saccharomyces cerevisiae. The two mutants are able to suppress the deletion of PAB1, and they both present a strong reduction in their 60S ribosomal subunit content. In an spb1-2 strain grown at the restrictive temperature, processing of the 27S pre-rRNA into mature 25S rRNA and 5.8S is completely abolished and production of mature 18S is reduced, while the abnormal 23S species is accumulated. Spb1p is a 96.5-kDa protein that is localized to the nucleolus. Coimmunoprecipitation experiments show that Spb1p is associated in vivo with the nucleolar proteins Nop1p and Nop5/58p. Protein sequence analysis reveals that Spb1p possesses a putative S-adenosyl-L-methionine (AdoMet)-binding domain, which is common to the AdoMet-dependent methyltransferases. We show here that Spb1p is able to bind [(3)H]AdoMet in vitro, suggesting that it is a novel methylase, whose possible substrates will be discussed.     

Sec7p directs the transitions required for yeast Golgi biogenesis

Endoplasmic reticulum (ER)-to-Golgi traffic in yeast proceeds by the maturation of membrane compartments from post-ER vesicles to intermediate small vesicle tubular clusters (VTCs) to Golgi nodular membrane networks (Morin-Ganet et al., Traffic 2000; 1: 56–68). The balance between ER and Golgi compartments is maintained by COPII- and COPI-mediated anterograde and retrograde traffic, which are dependent on Sec7p and ARF function. The sec7-4 temperature-sensitive allele is a mutation in the highly conserved Sec7 domain (Sec7d) found in all ARF-guanine nucleotide exchange factor proteins. Post-ER trafficking is rapidly inactivated in sec7-4 mutant yeast at the restrictive temperature. This conditional defect prevented the normal production of VTCs and instead generated Golgi-like tubes emanating from the ER exit sites. These tubes progressively developed into stacked cisternae defining the landmark sec7 mutant phenotype. Consistent with the in vivo results, a Sec7d peptide inhibited ER-to-Golgi transport and displaced Sec7p from its membrane anchor in vitro . The similarities in the consequences of inactivating Sec7p or ARFs in vivo was revealed by genetic disruption of yeast ARFs or by addition of brefeldin A (BFA) to whole cells. These treatments, as in sec7-4 yeast, affected the morphology of membrane compartments in the ER-Golgi transition. Further evidence for Sec7p involvement in the transition for Golgi biogenesis was revealed by in vitro binding between distinct domains of Sec7p with ARFs, COPI and COPII coat proteins. These results suggest that Sec7p coordinates membrane transitions in Golgi biogenesis by directing and scaffolding the binding and disassembly of coat protein complexes to membranes, both at the VTC transition from ER exit sites to form Golgi elements and for later events in Golgi maturation.     

Rsp1p, a J domain protein required for disassembly and assembly of microtubule organizing centers during the fission yeast cell cycle

Regulation of microtubule organizing centers (MTOCs) orchestrates the reorganization of the microtubule (MT) cytoskeleton. In the fission yeast Schizosaccharomyces pombe, an equatorial MTOC (eMTOC) at the cell division site disassembles after cytokinesis, and multiple interphase MTOCs (iMTOCs) appear on the nucleus. Here, we show that, upon eMTOC disassembly, small satellites carrying MTOC components such as the gamma-tubulin complex travel in both directions along interphase MTs. We identify rsp1p, an MTOC protein required for eMTOC disassembly. In rsp1 loss-of-function mutants, the eMTOC persists and organizes an abnormal microtubule aster, while iMTOCs and satellites are greatly reduced. Conversely, rsp1p overexpression inhibits eMTOC formation. Rsp1p is a J domain protein that interacts with an hsp70. Thus, our findings suggest a model in which rsp1p is part of a chaperone-based mechanism that disassembles the eMTOC into satellites, contributing to the dynamic redistribution of MTOC components for organization of interphase microtubules.     

Nop9 is an RNA binding protein present in pre-40S ribosomes and required for 18S rRNA synthesis in yeast

Proteomic analyses in yeast have identified a large number of proteins that are associated with preribosomal particles. However, the product of the yeast ORF YJL010C, herein designated as Nop9, failed to be identified in any previous physical or genetic analysis of preribosomes. Here we report that Nop9 is a nucleolar protein, which is associated with 90S and 40S preribosomes. In cells depleted of Nop9p, early cleavages of the 35S pre-rRNA are inhibited, resulting in the nucleolar retention of accumulated precursors and a failure to synthesize 18S rRNA. Nop9 contains multiple pumilio-like putative RNA binding repeats and displays robust in vitro RNA binding activity. The identification of Nop9p as a novel, essential factor in the nuclear maturation of 90S and pre-40S ribosomal subunits shows that the complement of ribosome synthesis factors remains incomplete.     

A novel genetic screen for snRNP assembly factors in yeast identifies a conserved protein, Sad1p, also required for pre-mRNA splicing

The assembly pathway of spliceosomal snRNPs in yeast is poorly understood. We devised a screen to identify mutations blocking the assembly of newly synthesized U4 snRNA into a functional snRNP. Fifteen mutant strains failing either to accumulate the newly synthesized U4 snRNA or to assemble a U4/U6 particle were identified and categorized into 13 complementation groups. Thirteen previously identified splicing-defective prp mutants were also assayed for U4 snRNP assembly defects. Mutations in the U4/U6 snRNP components Prp3p, Prp4p, and Prp24p led to disassembly of the U4/U6 snRNP particle and degradation of the U6 snRNA, while prp17-1 and prp19-1 strains accumulated free U4 and U6 snRNA. A detailed analysis of a newly identified mutant, the sad1-1 mutant, is presented. In addition to having the snRNP assembly defect, the sad1-1 mutant is severely impaired in splicing at the restrictive temperature: the RP29 pre-mRNA strongly accumulates and splicing-dependent production of beta-galactosidase from reporter constructs is abolished, while extracts prepared from sad1-1 strains fail to splice pre-mRNA substrates in vitro. The sad1-1 mutant is the only splicing-defective mutant analyzed whose mutation preferentially affects assembly of newly synthesized U4 snRNA into the U4/U6 particle. SAD1 encodes a novel protein of 52 kDa which is essential for cell viability. Sad1p localizes to the nucleus and is not stably associated with any of the U snRNAs. Sad1p contains a putative zinc finger and is phylogenetically highly conserved, with homologues identified in human, Caenorhabditis elegans, Arabidospis, and Drosophila.     

KCNQ1/KCNE1 assembly,co-translation not required

Voltage-gated potassium channels are often assembled with accessory proteins which increases their functional diversity. KCNE proteins are small accessory proteins that modulate voltage-gated potassium (K_V) channels. Although the functional effects of various KCNE proteins have been described, many questions remain regarding their assembly with the pore-forming subunits. For example, while previous experiments with some K_V channels suggest that the association of the pore-subunit with the accessory subunits occurs co-translationally in the endoplasmic reticulum, it is not known whether KCNQ1 assembly with KCNE1 occurs in a similar manner to generate the medically important cardiac slow delayed rectifier current (IKs). In this study we used a novel approach to demonstrate that purified recombinant human KCNE1 protein (prKCNE1) modulates KCNQ1 channels heterologously expressed in Xenopus oocytes resulting in generation of IKs. Incubation of KCNQ1-expressing oocytes with cycloheximide did not prevent IKs expression following prKCNE1 injection. By contrast, incubation with brefeldin A prevented KCNQ1 modulation by prKCNE1. Moreover, injection of the trafficking-deficient KCNE1-L51H reduced KCNQ1 currents. Together, these observations indicate that while assembly of KCNE1 with KCNQ1 does not require co-translation, functional KCNQ1-prKCNE1 channels assemble early in the secretory pathway and reach the plasma membrane via vesicular trafficking.     

Sgt1 is required for human kinetochore assembly

Budding yeast Sgt1 is required for kinetochore assembly, and its homologues have a role in cAMP signalling in fungi and pathogen resistance in plants. The function of mammalian Sgt1 is unknown. We report that RNA interference-mediated depletion of Sgt1 from HeLa cells causes dramatic alterations of the mitotic spindle and problems in chromosome alignment. Cells lacking Sgt1 undergo a mitotic delay due to activation of the spindle checkpoint. The checkpoint response, however, is significantly weakened in Sgt1-depleted cells, and this correlates with a dramatic reduction in kinetochore levels of Mad1, Mad2 and BubR1. These effects are explained by a problem in kinetochore assembly that prevents the localization of Hec1, CENP-E, CENP-F, CENP-I, but not CENP-C, to mitotic kinetochores. Our studies implicate Sgt1 as an essential protein and a critical assembly factor for the mammalian kinetochore, and lend credit to the hypothesis of a kinetochore assembly pathway that is conserved from yeast to man.