A role in folding of newly translated proteins in the cytosol of eukaryotes has been proposed for t-complex polypeptide-1 (TCP1), although its molecular targets have not yet been identified. Tubulin is a major cytosolic protein whose assembly into microtubules is critical to many cellular processes. Although numerous studies have focused on the expression of tubulin, little is known about the processes whereby newly translated tubulin subunits acquire conformations that enable them to form alpha-beta-heterodimers. We examined the biogenesis of alpha- and beta-tubulin in rabbit reticulocyte lysate, and report here that newly translated tubulin subunits entered a 900K complex in a protease-sensitive conformation. Addition of Mg-ATP, but not nonhydrolysable analogues, released the tubulin subunits as assembly-competent protein with a conformation that was relatively protease-resistant. The 900K complex purified from reticulocyte lysate contained as its major constituent a 58K protein that cross-reacted with a monoclonal antiserum against mouse TCP1. We conclude that TCP1 functions as a cytosolic chaperone in the biogenesis of tubulin.

Cleavage of amino-terminal octapeptides, F/L/IXXS/T/GXXXX, by mitochondrial intermediate peptidase (MIP) is typical of many mitochondrial precursor proteins imported to the matrix and the inner membrane.We previously described the molecular characterization of rat liver MIP (RMIP) and indicated a putative homolog in the sequence predicted from gene YCL57w of yeast chromosome III. A new yeast gene, MIPJ, has now been isolated by screening a Saccharomyces cerevisiae genomic library with an RMIP cDNA probe. MIP1 predicts a protein of 772 amino acids (YMIP), which is 54% similar and 31% identical to RMIP and includes a putative 37-residue mitochondrial leader peptide. RMIP and YMIP contain the sequence LFHEMGHAM HSMLGRT, which includes a zinc-binding motif, HEXXH, while the predicted YCL57w protein contains a comparable sequence with a lower degree of homology. No obvious biochemical phenotype was observed in a chromosomally disrupted ycl57w mutant. In contrast, a mipi mutant was unable to grow on nonfermentable substrates, while a mipi yclS7w double disruption did not result in a more severe phenotype. The mipi mutant exhibited defects of complexes III and IV of the respiratory chain, caused by failure to carry out the second MIP-catalyzed cleavage of the nuclear-encoded precursors for cytochrome oxidase subunit IV (CoxIV) and the iron-sulfur protein (Fe-S) of the bc, complex to mature proteins. Previous studies of Saccharomyces cerevisiae (6,12,15,22) and Neurospora crassa (17,51) suggest that the MIP-dependent pathway of mitochondrial enzyme maturation is conserved in lower eukaryotes. MIP MATERMILS AND METHODSYeast strains, growth media, and plasmids. The following previously described strains were used: YPH501 (MATa/MATat ura3-52/ura3-52 lys2-801ambber/lys2-801amnber ade2 lJ0Jochrelade2-j0jochre trpl-A63/trpl-A63 his3-A200/his3-A200 leu2-zI\/leu2-Al) (46), AW1-2 (MATa/MATot ura3-52/ura3-52 leu2-3,112/ leu2-3,112) (54), ot429c (AL4Tot mifl-1 his4-519 ura3-52 leu2-3,112 arg3) (35), and B35.1 (ALITot mif2-1 his4-519 ura3-52 leu2-3,112 arg3) (54). The following mutants were constructed in this study: mip] disruption strains Y6040, a YPH501 derivative (MA Tot ura3-52 lys2-801amber ade2 Jl0Jochr' trpl-.i63 his3-A200 leu2-AJ miplA::LEU2), and Y34, an AW1-2 derivative (MATot ura3-52 leu2-3,112 miplA::LEU2); yclS7w disruption strain Y191, an AW1-2 derivative (ALTa ura3-52 leu2-3,112 5603 on May 11, 2018 by guest

(22) and folding of newly translated tubulin (23). In CHO cells the TCP1 complex has been shown to bind newly translated actin and tubulin (25). In Saccharomyces cerevisiae, TCPJ has been shown to be an essential gene, and a cold-sensitive TCP1 mutant exhibited an abnormal appearance of the mitotic spindle (26), supporting a role in the biogenesis of tubulin.To further evaluate the function of the TCP1 complex, we have taken a combination of biochemical and genetic approaches utilizing S. cerevisiae. Here we report the primary structure$ of a second subunit of the complex, TCP1f3, and report on the phenotypes ofgene disruption and temperaturesensitive alleles of both TCPJ (termed here TCPla) and TCP (3. MATERIALS AND METHODSSequence Analysis. The insert of A clone 4950 from a S. cerevisiae chromosome IX library kindly supplied by Maynard Olson and Linda Riles (27) was sequenced (insert of 20,328 bp) after shotgun subcloning of size-selected fragments into M13mpl8 (28). Open reading frames were analyzed with the DIANA program (J. Crooke, T. S. Horsnell, and B.B., unpublished work).Epitope Tagging. Epitopes were added at the C-termini of TCP1a and TCP1(3 immediately upstream from the respective stop codons in coding sequences carried in centromeric (CEN) plasmids YCP50 and YCplac22 (29,30), in the case of a, the influenza hemagglutinin epitope YPYDVPDYA, and for A, the Myc epitope EQKLISEEDL. The hemagglutinin epitope was detected with mouse monoclonal antibody 12CA5 (31) supplied by Berkeley Antibody (Richmond, CA) with permission from Scripps Institute, and the Myc epitope was detected with ascites produced from the hybridoma cell line 9E10 (32), kindly supplied by J. Michael Bishop.Purification of TCP1 Complex from S. cerevisiae. Logarithmically growing cells were harvested and spheroplasts were prepared by zymolyase treatment (33). Spheroplasts were

Cleavage of amino-terminal octapeptides, F/L/IXXS/T/GXXXX, by mitochondrial intermediate peptidase (MIP) is typical of many mitochondrial precursor proteins imported to the matrix and the inner membrane. We previously described the molecular characterization of rat liver MIP (RMIP) and indicated a putative homolog in the sequence predicted from gene YCL57w of yeast chromosome III. A new yeast gene, MIP1, has now been isolated by screening a Saccharomyces cerevisiae genomic library with an RMIP cDNA probe. MIP1 predicts a protein of 772 amino acids (YMIP), which is 54% similar and 31% identical to RMIP and includes a putative 37-residue mitochondrial leader peptide. RMIP and YMIP contain the sequence LFHEMGHAM HSMLGRT, which includes a zinc-binding motif, HEXXH, while the predicted YCL57w protein contains a comparable sequence with a lower degree of homology. No obvious biochemical phenotype was observed in a chromosomally disrupted ycl57w mutant. In contrast, a mip1 mutant was unable to grow on nonfermentable substrates, while a mip1 ycl57w double disruption did not result in a more severe phenotype. The mip1 mutant exhibited defects of complexes III and IV of the respiratory chain, caused by failure to carry out the second MIP-catalyzed cleavage of the nuclear-encoded precursors for cytochrome oxidase subunit IV (CoxIV) and the iron-sulfur protein (Fe-S) of the bc1 complex to mature proteins. In vivo, intermediate-size CoxIV was accumulated in the mitochondrial matrix, while intermediate-size Fe-S was targeted to the inner membrane. Moreover, mip1 mitochondrial fractions failed to carry out maturation of the human ornithine transcarbamylase intermediate (iOTC), specifically cleaved by RMIP. A CEN plasmid-encoded YMIP protein restored normal MIP activity along with respiratory competence. Thus, YMIP is a functional homolog of RMIP and represents a new component of the yeast mitochondrial import machinery.

Despite significant strides in pre-emptive antiviral treatment of cytomegalovirus (CMV) infection after hematopoietic cell transplantation (HCT) and a corresponding reduction in CMV disease rates, toxicity from these therapies remains unacceptably high. New antivirals are urgently needed for CMV management. However, because of low CMV disease rates, clinical trials for new therapeutics and vaccines require large sample sizes to show clinical benefit.While data are mounting to support using CMV viral load (VL) as a surrogate endpoint in clinical trials and treatment protocols, a direct association between virologic markers and clinical endpoints from a placebo-controlled, randomized controlled trial (RCT) is lacking. Thus, we performed CMV DNA PCR testing on cryopreserved plasma samples collected during the first 100 days post-HCT in the only placebo-controlled, double-blind RCT of ganciclovir for the early treatment of CMV infection after HCT (Goodrich et al. NEJM 1991). In this landmark RCT, CMV disease and mortality were reduced dramatically in the first 180 days post-HCT. However, no quantitative surrogate markers were established. In the present study, viral kinetic parameters ( Figure 1A) were calculated from CMV DNA PCR values as continuous, timedependent variables. Cox proportional hazard models assessing associations between viral kinetic markers and time to CMV disease and death events were adjusted for donor CMV serostatus and acute graft-versus-host-disease, counting events to day 100 and day 180 post-HCT. Most recent VL, highest VL, and duration of viremia were strongly associated with time to CMV disease and time to the first event of CMV disease or death, suggesting that these markers may be useful as surrogate endpoints for these clinical outcomes (Table 1). In addition, we extended the survival analysis from the original report and found that mortality reduction in the ganciclovir arm was sustained to three years ( Figure 1B). Our results support using virologic markers as surrogates for CMV disease and death to facilitate delivery of potent, nontoxic CMV therapies to HCT recipients.