Diffusion-collision of foldons elucidates the kinetic effects of point mutations and suggests control strategies of the folding process of helical proteins

In this article we use mutation studies as a benchmark for a minimal model of the folding process of helical proteins. The model ascribes a pivotal role to the collisional dynamics of a few crucial residues (foldons) and predicts the folding rates by exploiting information drawn from the protein sequence. We show that our model rationalizes the effects of point mutations on the kinetics of folding. The folding times of two proteins and their mutants are predicted. Stability and location of foldons have a critical role as the determinants of protein folding. This allows us to elucidate two main mechanisms for the kinetic effects of mutations. First, it turns out that the mutations eliciting the most notable effects alter protein stability through stabilization or destabilization of the foldons. Secondly, the folding rate is affected via a modification of the foldon topology by those mutations that lead to the birth or death of foldons. The few mispredicted folding rates of some mutants hint at the limits of the current version of the folding model proposed in the present article. The performance of our folding model declines in case the mutated residues are subject to strong long-range forces. That foldons are the critical targets of mutation studies has notable implications for design strategies and is of particular interest to address the issue of the kinetic regulation of single proteins in the general context of the overall dynamics of the interactome.     

Unfolding and folding kinetics of amyotrophic lateral sclerosis-associated mutant Cu,Zn superoxide dismutases

More than 110 mutations in dimeric, Cu,Zn superoxide dismutase (SOD) have been linked to the fatal neurodegenerative disease, amyotrophic lateral sclerosis (ALS). In both human patients and mouse model studies, protein misfolding has been implicated in disease pathogenesis. A central step in understanding the misfolding/aggregation mechanism of this protein is the elucidation of the folding pathway of SOD. Here we report a systematic analyses of unfolding and folding kinetics using single- and double-jump experiments as well as measurements as a function of guanidium chloride, protein, and metal concentration for fully metallated (holo) pseudo wild-type and ALS-associated mutant (E100G, G93R, G93A, and metal binding mutants G85R and H46R) SODs. The kinetic mechanism for holo SODs involves native dimer, monomer intermediate, and unfolded monomer, with variable metal dissociation from the monomeric states depending on solution conditions. The effects of the ALS mutations on the kinetics of the holoproteins in guanidium chloride are markedly different from those observed previously for acid-induced unfolding and for the unmetallated (apo) forms of the proteins. The mutations decrease the stability of holo SOD mainly by increasing unfolding rates, which is particularly pronounced for the metal-binding mutants, and have relatively smaller effects on the observed folding kinetics. Mutations also seem to favour increased formation of a Zn-free monomer intermediate, which has been implicated in the formation of toxic aggregates. The results reveal the kinetic basis for the extremely high stability of wild-type holo SOD and the possible consequences of kinetic changes for disease.     

Role of electrostatic interactions for the stability and folding behavior of cold shock protein

The impacts of three charged‐residue‐involved mutations, E46A, R3E, and R3E/L66E, on the thermostability and folding behavior of the cold shock protein from the themophile Bacillus caldolyticus (Bc‐Csp) were investigated by using a modified Gō‐like model, in which the nonspecific electrostatic interactions of charged residues were taken into account. Our simulation results show that the wild‐type Bc‐Csp and its three mutants are all two‐sate folders, which is consistent with the experimental observations. It is found that these three mutations all lead to a decrease of protein thermodynamical stability, and the effect of R3E mutation is the strongest. The lower stability of these three mutants is due to the increase of the enthalpy of the folded state and the entropy of the unfolded state. Using this model, we also studied the folding kinetics and the folding/unfolding pathway of the wild‐type Bc‐Csp as well as its three mutants and then discussed the effects of electrostatic interactions on the folding kinetics. The results indicate that the substitutions at positions 3 and 46 largely decrease the folding kinetics, whereas the mutation of residue 66 only slightly decreases the folding rate. This result agrees well with the experimental observations. It is also found that these mutations have little effects on the folding transition state and the folding pathway, in which the N‐terminal β sheet folds earlier than the C‐terminal region. We also investigated the detailed unfolding pathway and found that it is really the reverse of the folding pathway, providing the validity of our simulation results. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Experimental Evolution of Adenylate Kinase Reveals Contrasting Strategies toward Protein Thermostability

Success in evolution depends critically upon the ability of organisms to adapt, a property that is also true for the proteins that contribute to the fitness of an organism. Successful protein evolution is enhanced by mutational pathways that generate a wide range of physicochemical mechanisms to adaptation. In an earlier study, we used a weak-link method to favor changes to an essential but maladapted protein, adenylate kinase (AK), within a microbial population. Six AK mutants (a single mutant followed by five double mutants) had success within the population, revealing a diverse range of adaptive strategies that included changes in nonpolar packing, protein folding dynamics, and formation of new hydrogen bonds and electrostatic networks. The first mutation, AK_BSUB Q199R, was essential in defining the structural context that facilitated subsequent mutations as revealed by a considerable mutational epistasis and, in one case, a very strong dependence upon the order of mutations. Namely, whereas the single mutation AK_BSUB G213E decreases protein stability by >25°C, the same mutation in the background of AK_BSUB Q199R increases stability by 3.4°C, demonstrating that the order of mutations can play a critical role in favoring particular molecular pathways to adaptation. In turn, protein folding kinetics shows that four of the five AK_BSUB double mutants utilize a strategy in which an increase in the folding rate accompanied by a decrease in the unfolding rate results in additional stability. However, one mutant exhibited a dramatic increase in the folding relative to a modest increase in the unfolding rate, suggesting a different adaptive strategy for thermostability. In all cases, an increase in the folding rates for the double mutants appears to be the preferred mechanism in conferring additional stability and may be an important aspect of protein evolution. The range of overlapping as well as contrasting strategies for success illustrates both the power and subtlety of adaptation at even the smallest unit of change, a single amino acid.     

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates from Korea and analysis of the correlation between the mutations and pyrazinamidase activity

To investigate the effect of natural pyrazinamidase (PncA) mutations on protein function, we analyzed expression and PncA activity of eight pncA point mutants identified in nineteen pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates. Among them, two mutants (Y99D and T135P) showed high expression level and solubility comparable to those of the wild-type PncA protein, two (K48E and G97D) displayed low expression level and solubility, and four (C14R, H51P, W68S, and A146V) were insoluble. Interestingly, when possible structural effects of these mutations were predicted by the CUPSAT program based on the proposed three-dimensional structure of M. tuberculosis PncA, only two highly soluble mutant proteins (Y99D and T135P) were predicted to be stabilizing and have favorable torsion angles. However, the others exhibiting either low solubility or precipitation were foreseen to be destabilizing and/or have unfavorable torsion angles, suggesting that the alterations could interfere with proper protein folding, thereby decreasing or depleting protein solubility. A PncA activity assay demonstrated that two mutants (G97D and T135P) showed virtually no activity, but two other mutants (K48E and Y99D) exhibited wild-type activity, indicating that the PncA residues (Cys14, His51, Trp68, Gly97, Thr135, and Ala146) may be important for PncA activity and/or proper protein folding.     

Mutational analysis of the folding transition state of the C-terminal domain of ribosomal protein L9: a protein with an unusual beta-sheet topology

The C-terminal domain of ribosomal protein L9 (CTL9) is a 92-residue alpha-beta protein which contains an unusual three-stranded mixed parallel and antiparallel beta-sheet. The protein folds in a two-state fashion, and the folding rate is slow. It is thought that the slow folding may be caused by the necessity of forming this unusual beta-sheet architecture in the transition state for folding. This hypothesis makes CTL9 an interesting target for folding studies. The transition state for the folding of CTL9 was characterized by phi-value analysis. The folding of a set of hydrophobic core mutants was analyzed together with a set of truncation mutants. The results revealed a few positions with high phi-values (> or = 0.5), notably, V131, L133, H134, V137, and L141. All of these residues were found in the beta-hairpin region, indicating that the formation of this structure is likely to be the rate-limiting step in the folding of CTL9. One face of the beta-hairpin docks against the N-terminal helix. Analysis of truncation mutants of this helix confirmed its importance in folding. Mutations at other sites in the protein gave small phi-values, despite the fact that some of them had major effects on stability. The analysis indicates that formation of the antiparallel hairpin is critical and its interactions with the first helix are also important. Thus, the slow folding is not a consequence of the need to fully form the unusual three-stranded beta-sheet in the transition state. Analysis of the urea dependence of the folding rates indicates that mutations modulate the unfolded state. The folding of CTL9 is broadly consistent with the nucleation-condensation model of protein folding.     

Mechanism of phage P22 tailspike protein folding mutations.

Temperature-sensitive folding (tsf) and global-tsf-suppressor (su) point mutations affect the folding yields of the trimeric, thermostable phage P22 tailspike endorhamnosidase at elevated temperature, both in vivo and in vitro, but they have little effect on function and stability of the native folded protein. To delineate the mechanism by which these mutations modify the partitioning between productive folding and off-pathway aggregation, the kinetics of refolding after dilution from acid-urea solutions and the thermal stability of folding intermediates were analyzed. The study included five tsf mutations of varying severity, the two known su mutations, and four tsf/su double mutants. At low temperature (10 degrees C), subunit-folding rates, measured as an increase in fluorescence, were similar for wild-type and mutants. At 25 degrees C, however, tsf mutations reduced the rate of subunit folding. The su mutations increased this rate, when present in the tsf-mutant background, but had no effect in the wild-type background. Conversely, tsf mutations accelerated, and su mutations retarded the irreversible off-pathway reaction, as revealed by temperature down-shifts after varied times during refolding at high temperature (40 degrees C). The kinetic results are consistent with tsf mutations destabilizing and su mutations stabilizing an essential subunit folding intermediate. In accordance with this interpretation, tsf mutations decreased, and su mutations increased the temperature resistance of folding intermediates, as disclosed by temperature up-shifts during refolding at 25 degrees C. The stabilizing and destabilizing effects were most pronounced early during refolding. However, they were not limited to subunit-folding intermediates and were also observable during thermal unfolding of the native protein.     

Structural requirements for the stability and microsomal transport activity of the human glucose 6-phosphate transporter

Deficiencies in glucose 6-phosphate (G6P) transporter (G6PT), a 10-helical endoplasmic reticulum transmembrane protein of 429 amino acids, cause glycogen storage disease type 1b. To date, only three missense mutations in G6PT have been shown to abolish microsomal G6P transport activity. Here, we report the results of structure-function studies on human G6PT and demonstrate that 15 missense mutations and a codon deletion (delta F93) mutation abolish microsomal G6P uptake activity and that two splicing mutations cause exon skipping. While most missense mutants support the synthesis of G6PT protein similar to that of the wild-type transporter, immunoblot analysis shows that G20D, delta F93, and I278N mutations, located in helix 1, 2, and 6, respectively, destabilize the G6PT. Further, we demonstrate that G6PT mutants lacking an intact helix 10 are misfolded and undergo degradation within cells. Moreover, amino acids 415-417 in the cytoplasmic tail of the carboxyl-domain, extending from helix 10, also play a critical role in the correct folding of the transporter. However, the last 12 amino acids of the cytoplasmic tail play no essential role(s) in functional integrity of the G6PT. Our results, for the first time, elucidate the structural requirements for the stability and transport activity of the G6PT protein.     

Chemical chaperones assist intracellular folding to buffer mutational variations

Hidden genetic variations have the potential to lead to the evolution of new traits. Molecular chaperones, which assist protein folding, may conceal genetic variations in protein-coding regions. Here we investigate whether the chemical milieu of cells has the potential to alleviate intracellular protein folding, a possibility that could implicate osmolytes in concealing genetic variations. We found that the model osmolyte trimethylamine N-oxide (TMAO) can buffer mutations that impose kinetic traps in the folding pathways of two model proteins. Using this information, we rationally designed TMAO-dependent mutants in vivo, starting from a TMAO-independent protein. We show that different osmolytes buffer a unique spectrum of mutations. Consequently, the chemical milieu of cells may alter the folding pathways of unique mutant variants in polymorphic populations and lead to unanticipated spectra of genetic buffering.     

Identification of amino acids important for the biochemical activity of Methanothermobacter thermautotrophicus MCM

Methanothermobacter thermautotrophicus minichromosomal maintenance protein (mtMCM) is a 75 kDa protein that self-assembles into a double hexamer structure. The double hexamer formed by the N-terminal region of mtMCM has a highly charged (overwhelmingly net positive) inner channel. Here we investigate the effects of point mutations of some of these charged residues on the biological activities of mtMCM. Although all of the mutants were similar to the wild type in protein folding and complex assembly, we found that mutations impaired helicase activity. The study of the DNA binding and ATPase activities of these mutants revealed that the impairment of the helicase activity was highly correlated with a decrease in DNA binding, providing evidence consistent with the role of these charged residues of the inner channel in interactions with DNA.     

Intragenic Suppressing Mutations Correct the Folding and Intracellular Traffic of Misfolded Mutants of Yor1p,a Eukaryotic Drug Transporter

ATP-binding cassette (ABC) transporters play pivotal physiological roles in substrate transport across membranes, and defective assembly of these proteins can cause severe disease associated with improper drug or ion flux. The yeast protein Yor1p is a useful model to study the biogenesis of ABC transporters; deletion of a phenylalanine residue in the first nucleotide-binding domain (NBD1) causes misassembly and retention in the endoplasmic reticulum (ER) of the resulting protein Yor1p-ΔF670, similar to the predominant disease-causing allele in humans, CFTR-ΔF508. Here we describe two novel Yor1p mutants, G278R and I1084P, which fail to assemble and traffic similar to Yor1p-ΔF670. These mutations are located in the two intracellular loops (ICLs) that interface directly with NBD1, and thus disrupt a functionally important structural module. We isolated 2 second-site mutations, F270S and R1168M, which partially correct the folding injuries associated with the G278R, I1084P, and ΔF670 mutants and reinstate their trafficking. The position of both corrective mutations at the cytoplasmic face of a transmembrane helix suggests that they restore biogenesis by influencing the behavior of the transmembrane domains rather than by direct restoration of the ICL1-ICL4-NBD1 structural module. Given the conserved topology of many ABC transporters, our findings provide new understanding of functionally important inter-domain interactions and suggest new potential avenues for correcting folding defects caused by abrogation of those domain interfaces.     

Role of the extracellular loop in the folding of a CFTR transmembrane helical hairpin

The folding of membrane-spanning domains into their native functional forms depends on interactions between transmembrane (TM) helices joined by covalent loops. However, the importance of these covalent linker regions in mediating the strength of helix-helix associations has not been systematically addressed. Here we examine the potential structural impact of cystic fibrosis-phenotypic mutations in the extracellular loop 2 (ECL2) on interactions between the TM3 and TM4 helices of the cystic fibrosis transmembrane conductance regulator (CFTR) in constructs containing CFTR residues 194-241. When the effects of replacements in ECL2 (including the CF-phenotypic mutants E217G and Q220R) were evaluated in a library of wild-type and mutant TM3-ECL2-TM4 hairpin constructs, we found that SDS-PAGE gel migration rates differed over a range of nearly 40% +/- the wild-type position and that decreased migration rates correlate with increasing hairpin alpha-helical content as measured by circular dichroism spectra in sodium dodecyl sulfate micelles. The decreased mobility of TM3/4 constructs by introduction of non-native residues is interpreted in terms of an elongation or "opening" of the helical hairpin and concomitant destabilization of membrane-based helix-helix interactions. Our results support a role for short loop regions in dictating the stability of membrane protein folds and highlight the interplay between membrane-embedded helix-helix interactions and loop conformation in influencing the structure of membrane proteins.     

All-Atom Simulations Reveal How Single-Point Mutations Promote Serpin Misfolding

Protein misfolding is implicated in many diseases, including serpinopathies. For the canonical inhibitory serpin α₁-antitrypsin, mutations can result in protein deficiencies leading to lung disease, and misfolded mutants can accumulate in hepatocytes, leading to liver disease. Using all-atom simulations based on the recently developed bias functional algorithm, we elucidate how wild-type α₁-antitrypsin folds and how the disease-associated S (Glu264Val) and Z (Glu342Lys) mutations lead to misfolding. The deleterious Z mutation disrupts folding at an early stage, whereas the relatively benign S mutant shows late-stage minor misfolding. A number of suppressor mutations ameliorate the effects of the Z mutation, and simulations on these mutants help to elucidate the relative roles of steric clashes and electrostatic interactions in Z misfolding. These results demonstrate a striking correlation between atomistic events and disease severity and shine light on the mechanisms driving chains away from their correct folding routes.     

Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of conserved glycine residues and the identification of additional conserved protein motifs in eNTPDases.

Glycine residues are recognized as important structural determinants in nucleotide-binding domains of many enzymes. The functional significance of seven glycine residues invariant in all 22 eNTPDase sequences was therefore examined. Glycine-to-alanine mutants of eNTPDase3 were analyzed for nucleotidase activities and tertiary and quaternary structure changes. Mutations G98A and G183A had modest effects on ATPase and ADPase activities. The G141A mutation resulted in 4- to 5-fold decreased nucleotidase activity, while the G222A mutation decreased ATPase activity 20-fold, and ADPase activity 6-fold. Unlike the other five glycine mutants, the G263A and G462A mutations caused significant loss of nucleotidase activity which was observed concomitant with lower protein expression levels, large-scale changes in tertiary and quaternary protein structure, and decreased trafficking to the plasma membrane. Thus, these data identify glycine residues that are essential for enzymatic activity and the tertiary and quaternary structure of eNTPDase3. Further, two additional conserved regions in the eNTPDases are identified, apyrase conserved regions ACR1a and ACR4a, which may be involved in phosphate binding/hydrolysis and protein folding, respectively.     

Kinetics and energetics of the translocation of maltose binding protein folding mutants

Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB.     

An on-pathway hidden intermediate and the early rate-limiting transition state of Rd-apocytochrome b562 characterized by protein engineering

The folding pathway of Rd-apocytochrome b562, a four-helix bundle protein, was characterized using Trp and Ala/Gly pair mutations. We found that the Trp mutants (F65W) of both the fully folded Rd-apocytochrome b562 and a partially unfolded intermediate with the N-terminal helix (helix I) unfolded, fold with identical folding rates, providing direct evidence for the conclusion that the rate-limiting transition state folds before the partially unfolded intermediate; and that this hidden intermediate is an on-pathway intermediate. We further characterized the helical structures formed in the rate-limiting transition state by measuring the folding/unfolding rates for Ala/Gly pair mutations at solvent-exposed positions. Little change in folding rates occurred for the Ala/Gly pair mutations at positions in helix I and the C-terminal regions of helix II and IV. In contrast, a significant difference in folding rates was observed for the Ala/Gly pair mutations in helix III and the N-terminal regions of helix II and IV, suggesting that helix III and the N-terminal regions of helix II and IV are formed in the rate-limiting transition state. These results complement those obtained from earlier studies and help to define the folding pathway of Rd-apocytochrome b562 in more detail.     

The Limited Role of Nonnative Contacts in the Folding Pathways of a Lattice Protein

Models of protein energetics that neglect interactions between amino acids that are not adjacent in the native state, such as the Gō model, encode or underlie many influential ideas on protein folding. Implicit in this simplification is a crucial assumption that has never been critically evaluated in a broad context: Detailed mechanisms of protein folding are not biased by nonnative contacts, typically argued to be a consequence of sequence design and/or topology. Here we present, using computer simulations of a well-studied lattice heteropolymer model, the first systematic test of this oft-assumed correspondence over the statistically significant range of hundreds of thousands of amino acid sequences that fold to the same native structure. Contrary to previous conjectures, we find a multiplicity of folding mechanisms, suggesting that Gō-like models cannot be justified by considerations of topology alone. Instead, we find that the crucial factor in discriminating among topological pathways is the heterogeneity of native contact energies: The order in which native contacts accumulate is profoundly insensitive to omission of nonnative interactions, provided that native contact heterogeneity is retained. This robustness holds over a surprisingly wide range of folding rates for our designed sequences. Mirroring predictions based on the principle of minimum frustration, fast-folding sequences match their Gō-like counterparts in both topological mechanism and transit times. Less optimized sequences dwell much longer in the unfolded state and/or off-pathway intermediates than do Gō-like models. For dynamics that bridge unfolded and unfolded states, however, even slow folders exhibit topological mechanisms and transit times nearly identical with those of their Gō-like counterparts. Our results do not imply a direct correspondence between folding trajectories of Gō-like models and those of real proteins, but they do help to clarify key topological and energetic assumptions that are commonly used to justify such caricatures.     

ALS-linked FUS mutations dysregulate G-quadruplex-dependent liquid–liquid phase separation and liquid-to-solid transition

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the accumulation of protein aggregates in motor neurons. Recent discoveries of genetic mutations in ALS patients promoted research into the complex molecular mechanisms underlying ALS. FUS (fused in sarcoma) is a representative ALS-linked RNA-binding protein (RBP) that specifically recognizes G-quadruplex (G4)-DNA/RNAs. However, the effects of ALS-linked FUS mutations on the G4-RNA-binding activity and the phase behavior have never been investigated. Using the purified full-length FUS, we analyzed the molecular mechanisms of multidomain structures consisting of multiple functional modules that bind to G4. Here we succeeded to observe the liquid–liquid phase separation (LLPS) of FUS condensate formation and subsequent liquid-to-solid transition (LST) leading to the formation of FUS aggregates. This process was markedly promoted through FUS interaction with G4-RNA. To further investigate, we selected a total of eight representative ALS-linked FUS mutants within multidomain structures and purified these proteins. The regulation of G4-RNA-dependent LLPS and LST pathways was lost for all ALS-linked FUS mutants defective in G4-RNA recognition tested, supporting the essential role of G4-RNA in this process. Noteworthy, the P525L mutation that causes juvenile ALS exhibited the largest effect on both G4-RNA binding and FUS aggregation. The findings described herein could provide a clue to the hitherto undefined connection between protein aggregation and dysfunction of RBPs in the complex pathway of ALS pathogenesis.     

Suppressor mutations in the transmembrane segments of P-glycoprotein promote maturation of processing mutants and disrupt a subset of drug-binding sites

Defective folding of cystic fibrosis transmembrane conductance regulator protein missing Phe508 (DeltaF508) is the major cause of cystic fibrosis. The folding defect in DeltaF508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (DeltaY490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R). Possible mechanisms of arginine rescue were that they mimicked some of the effects of drug substrate interactions with P-gp or that they affected global folding such that all drug substrate/modulator interactions with P-gp were altered. To distinguish between these mechanisms, we tested whether arginines introduced into other TMs predicted to line the drug-binding pocket (TM1 or TM3) would affect folding. It was found that mutation of L65R(TM1) or T199R(TM3) promoted maturation of processing mutants. We then tested whether arginine suppressor mutations had local or global effects on P-gp interactions with drug substrates and modulators. The L65R(TM1), T199R(TM3), I306R(TM5), or F343R(TM6) mutations were introduced into the P-gp mutant L339C(TM6)/F728C(TM7), and thiol cross-linking was carried out in the presence of various concentrations of vinblastine, cyclosporin A, or rhodamine B. The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold. These results show that the arginine mutations affect a subset of drug-binding sites and suggest that they rescue processing mutants by mimicking drug substrate interactions with P-gp.     

Protein synthesis and the presence or absence of a measurable G1 in cultured chinese hamster cells

V79-8 cells lack a measurable G1 interval under normal growth conditions. We found that partial inhibition of protein synthesis using low levels of cycloheximide (0.05 μ/ml) could induce a measurable G1 in these cells without any significant effects on S, G2, or M. In view of these findings, recessive mutants selected from the V79-8 cell line, which each express G1, were analyzed for their rates of protein synthesis and degradation/loss. Three of the four mutants showed a decreased rate of protein synthesis sufficient to account for their G1 lengths. A fourth mutant, however, showed parental rates of both protein synthesis and degradation/loss. These results suggest not only that a G1 interval can be expressed as a result of a decreased rate of protein synthesis, but that other alterations (mutations) other than those simply affecting overall protein synthesis can result in the expression of a measureable G1 interval.