Analysis of the pH-dependent folding and stability of histidine point mutants allows characterization of the denatured state and transition state for protein folding

pH-Dependent studies of the folding kinetics and stability of a set of His to Gln point mutants were used to characterize the denatured state and transition state ensembles for the C-terminal domain of the ribosomal protein L9 (CTL9). CTL9 contains three histidine residues, two of which, H106 and H134, are buried in the native state, while the third, H144, is more exposed. Comparison of the pH-dependent stability calculated using the Tanford-Wyman linkage relationship to the measured values demonstrates that the apparent pK(a) values of the three histidine residues are not significantly perturbed in the denatured state ensemble. Kinetic measurements show that mutation of H134 has a larger effect on the folding process than does mutation of H106 and H144. The Phi-value for H134 is significantly larger than the Phi-values for the other histidine residues, which are near zero at both pH 5.45 and pH 8.0. The Phi-value for H134 is higher, 0.55, at pH 8.0 than at pH 5.45, 0.39. At pH 5.45, H134 is protonated in the unfolded state but deprotonated in the native state, while at pH 8.0 it is deprotonated in both. There is an excellent linear relationship between stability (logK) and folding rates (logk(f)) over the range of pH 5-9 for all mutants. From these plots, the ratio of DeltaQ( not equal)/DeltaQ can be calculated for each mutant. DeltaQ( not equal) is the difference in the number of protons bound to the transition state and to the unfolded state, while DeltaQ represents the difference between folded and denatured state. The linear plots indicate that the relative position of the transition state ensemble as judged by DeltaQ( not equal)/DeltaQ is independent of pH. The linkage analysis is consistent with the Phi-value analysis, showing that H134 is the most critical contributor to the development of pH-dependent interactions, including desolvation effects in the transition state ensemble.     

ADP Ribosylation Factor 1 Is Required for Synaptic Vesicle Budding in PC12 Cells

Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein–T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O''Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041–1049). A peptide spanning the effector domain of human ARF1 (2–17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.Generation of carrier vesicles from plasma membrane or intracellular membranous compartments involves at least two components, GTP-binding proteins and coats (Rothman and Wieland, 1996; Schekman and Orci, 1996). A particularly widespread protein that regulates coat assembly on intracellular membranes is ADP ribosylation factor (ARF)¹ 1, a small GTP-binding protein (Donaldson and Klausner, 1994; Boman and Kahn, 1995). The budding of vesicles from Golgi cisternae can be fully reconstituted in the presence of ARF1 and coatomer (COPI) (Ostermann et al., 1993; Donaldson and Klausner, 1994; Boman and Kahn, 1995; Rothman and Wieland, 1996). ARF1 recruits coatomers to the budding vesicle and couples uncoating to fusion with target membranes (Ostermann et al., 1993; Tanigawa et al., 1993). ARF1 is also required for the recruitment of COPI to vesicles budding from the ER (Bednarek et al., 1995). A hallmark of ARF1-mediated processes is their sensitivity to the fungal metabolite brefeldin A (BFA).The GDP–GTP exchange activity that replaces GDP bound to ARF proteins with GTP is inhibited by BFA (Donaldson et al., 1992; Helms and Rothman, 1992). The GDP form of ARF1 is unable to bind membranes and consequently, to recruit coats (Robinson and Kreis, 1992; Donaldson and Klausner, 1994). The selectivity of BFA is such that, if a membrane traffic event is sensitive to BFA, it is predicted to require ARF proteins. Inhibition of intra-Golgi and ER to Golgi traffic by BFA probably involves the COPI coatomers. BFA also interferes with coats other than COPI, especially those involved in budding from TGN. Thus it inhibits the formation of vesicles from the TGN (Simon et al., 1996) and causes the redistribution of assembly protein 1 and clathrin to the cytosol (Robinson and Kreis, 1992). Post-Golgi trafficking of the mannose-6-phosphate receptor (Wood et al., 1991) and the maturation of secretory granules (Dittie et al., 1996) are also sensitive to BFA. In addition to clathrin and COPI, BFA affects the recruitment of other “coating” molecules, such as the p47–βNAP complex (Simpson et al., 1996; Dell''Angelica et al., 1997) and p200 (Narula and Stow, 1995) to TGN membranes.Some endocytotic pathways are also sensitive to BFA. For example, the delivery of polyimmunoglobulin A (pIgA) to plasma membrane from the specialized apical endosome in epithelial MDCK cells, or from dendritic endosomes in hippocampal neurons, is inhibited by BFA (Hunziker et al., 1991; Barroso and Sztul, 1994). BFA-sensitive recruitment of COP1-related proteins and ARF proteins to endosomes has also been reported (Whitney et al., 1995; Cavenagh et al., 1996).The formation of synaptic vesicles at nerve terminals is a specialized endocytotic pathway that has many similarities to the formation of carrier vesicles from Golgi membranes. In this case, the donor membrane for synaptic vesicle formation is the plasma membrane or the endosome (De Camilli and Takei, 1996). Morphological evidence strongly suggests that synaptic vesicles are generated in nerve terminals through a coat-dependent mechanism (Shupliakov et al., 1997). In lysed nerve terminals, recruitment of dynamin and clathrin coats to membranous organelles is modulated by nonhydrolyzable GTP analogues (Takei et al., 1996). Cell-free reconstitution assays of neuroendocrine synaptic vesicle (SV) formation in PC12 cell extracts showed that GTPγS blocks the generation of properly sized SVs (Desnos et al., 1995), but the identity of the GTP-binding protein or proteins was not determined.In this paper, we show that reagents that interfere with the cycling of ARF1 between cytosol and membranes block SV formation in neuroendocrine PC12 cells. SV formation was reconstituted in vitro using recombinant ARF1 and a cytosol-derived high molecular weight fraction. Since SV production in vitro is from an endocytotic pool, these results suggest that coating mechanisms associated with ER and Golgi biosynthetic pathways are also associated with at least one endocytotic pathway.     

Partial amino acid sequence and genetic control of latent a2 allotype induced in rabbits by immunization with anti-a2 antibody

We have shown that after immunization of homozygous a1 rabbits of the B immunoglobulin (Ig) heavy chain haplotype with anti-a2 antibody (Ab) a population of molecules appears that has all of the serologic characteristics of the a2 allotype. We have now isolated these putative latent a2 molecules, have separated the heavy chains, and after enzymatic deblocking, have determined the first 19 N-terminal amino acids. For all eight allotype-associated residues, these putative latent a2 molecules have the amino acid residues typical of a2 allotype. As expected, the preimmune IgG from this a1a1 rabbit has the amino acids typical of the a1 allotype. Thus by partial amino acid sequence analysis, we provide additional evidence that the latent a2 allotype can be induced in a1a1 rabbits of the B heavy chain haplotype by immunization with anti-a2 Ab. Rabbits of other heavy chain haplotypes were also immunized with anti-a2 Ab and were tested for their ability to synthesize latent a2 allotype. Thus far, a1a1 rabbits of the A, B, C, and I heavy chain haplotypes all synthesize latent a2 allotype. In contrast, a3a3 rabbits of the G and H heavy chain haplotypes did not synthesize latent a2 allotype.     

Lethal and mutagenic effects of radiation and alkylating agents on two strains of mouse L5178Y cells

In previous studies, the two closely related strains of L5178Y (LY) mouse lymphoma cells, LY-R and LY-S, have been shown to differ in their sensitivity to UV and ionizing radiation. Thus, in comparison to strain LY-R, strain LY-S has been found to be more sensitive to the lethal effects of ionizing radiation, more resistant to the lethal effects of UV radiation, but less mutable at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus by both UV and X-radiation. In the present work, the lethal and mutagenic effects of ethyl methanesulfonate (EMS), methyl nitrosourea (MNU) and UV radiation (254 nm) were compared in the two strains. Mutability at the Na+/K+-ATPase locus as well as the HGPRT locus was determined. As previously reported, we found strain LY-S to be more resistant than strain LY-R to the lethal effects of UV radiation. In contrast, strain LY-S was more sensitive to the cytotoxic effects of the two alkylating agents. In spite of these differences in sensitivity, we found strain LY-S to be less mutable than strain LY-R by all 3 agents at the HGPRT locus. At the Na+/K+-ATPase locus, strain LY-S was also less mutable than strain LY-R by equal concentrations of EMS and UV radiation and by equitoxic concentrations of MNU. However, the difference between the strains was much more pronounced at the HGPRT locus than at the Na+/K+-ATPase locus. We have suggested that the interaction of unrepaired lesions in strain LY-S tends to cause an excess of deletions and multilocus effects, which in turn result in a locus-dependent decrease in the recovery of viable LY-S mutant cells.     

Male-specific DNA sequences in pigs

One hundred primers (Operon kits OPAA, OPAO, OPAV, OPC, and OPE series) were used for random amplified polymorphic DNA (RAPD) fingerprinting to determine male-specific fragments. Seventy-four percent of the primers yielded Yorkshire polymorphic fragments. One of these primers, OPAV-18, produced a novel 1098-bp DNA fragment found only in tested males. This male-specific fragment was isolated and constructed into plasmids for nucleotide sequencing. Two primers (5'-TTGCTCACGG TAGATAACAA GAGAG-3' and 5'-TTGCTCACGG ACCAGGTAGG GAATG-3') were designed according to the cloned male-specific sequence to amplify the male-specific band using polymerase chain reaction (PCR) for pig sexing. Sex-specific bands in the PCR gel products were represented in males but none were found in females when Yorkshire, Duroc, and Landrace genomic DNA samples were amplified with these two primers by PCR. The PCR products in the gel were transferred to nylon membranes and hybridized with a 32P-dCTP labeled probe of the cloned male-specific DNA fragment. There was a clear hybridization signal in samples from all of the male pigs, but not from those of female pigs. Male and female genomic DNA samples from these pigs were spotted onto nylon membranes and hybridized with the male-specific probe. The probe hybridized strongly to males only. A high degree of sequence homology was found among the novel male-specific DNA sequences in Yorkshire, Duroc and Landrace. The sex of these three breeds of pigs could be easily and effectively determined using these two primers.     

The control of mitochondrial respiration in yeast: A possible role of the outer mitochondrial membrane

Mitochondrial respiration in yeast (S. cerevisiae) is regulated by the level of glucose in the medium. Glucose is known to inhibit respiration by repressing key enzymes in the respiratory chain. We present evidence that the early events in this inhibition include the closure of VDAC channels, the primary pathway for metabolite flow across the outer membrane. Aluminum hydroxide is known to inhibit the closure of VDAC. Addition of aluminum acetylacetonate to yeast cells, which should elevate the aluminum hydroxide concentrations in the cytoplasm, caused the inhibition of cell respiration by glucose to be delayed for up to 100 min. No significant effect of aluminum was observed in cells grown on glycerol. Yeast cells lacking the VDAC gene were also unresponsive to the addition of aluminum salt in the presence of glucose. Therefore, the closure of VDAC channels may be an early step in the inhibition of the respiration of yeast by glucose.     

Mutagenesis and transformation of C3H 10T12 cells treated with ethyl methanesulfonate

Survival, mutagenesis and transformation were measured in mouse embryo C3H $\text{10}\text{T}\text{1}\text{2}$ cells following treatment with ethyl methanesulfonate (EMS). Ouabain-resistant cells and transformed cells were isolated, and reconstruction experiments were carried out to determine the optimum conditions for the measurement of mutation and transformation frequencies. Survival was measured by plating efficiency; mutagenesis was measured in terms of the induction of cells able to form colonies in the presence of ouabain; and transformation was measured by the induction of cells forming either morphologically altered colonies on a monolayer of contact-inhibited cells or of cells capable of forming colonies in semi-solid media. When confluent monolayers were incubated for 4 h after treatment with EMS, to allow excision repair before the resumption of DNA synthesis, survival as well as the frequencies of both mutation and transformation increased. When this repair (or holding) period was extended to 24 h, the frequencies of mutation and transformation both decreased as compared to the 4-h holding period. Thus, the holding periods affect the frequencies of EMS-induced mutagenesis and transformation similarly.