Further studies on a mutant mammalian cell line defective in mitosis

Experiments have been performed on a temperature-sensitive hamster cell line, ts-546. After the cells are switched to the non-permissive temperature, interphase cells continue through the cell cycle until the cells enter metaphase. Normal mitotic events then fail to occur. Metaphase chromosomes in the cells condense and coalesce into chromatin aggregates. Nuclear membrane re-forms around the aggregates resulting in the formation of mono-, bi- or multi-nucleate interphase-like cells. The conversion of mitotic cells to interphase-like states is completed within a few hours. The initial characterization of the mutant cell line was based on the observation that rounded-up cells accumulate in culture at the non-permissive temperature. The mitotic roundingup process may be utilized as a useful marker for selective isolation of mutant cell lines defective in mitosis.     

In vivo and in vitro analysis of ptl1, a yeast ts mutant with a membrane-associated defect in protein translocation.

Mutants defective in the ability to translocate proteins across the membrane of the endoplasmic reticulum were selected in Trp- Saccharomyces cerevisiae on the basis of their ability to retain a fusion protein in the cytosol. The fusion comprised the prepro region of prepro-alpha-factor (MF alpha 1) N-terminal to phosphoribosyl anthranilate isomerase (TRP1). The first of the protein translocation mutations, called ptl1, results in temperature-sensitivity of growth and protein translocation. At the non-permissive temperature, precursors to several secretory proteins accumulate in the cytosol. Using this mutant, we demonstrate that the prepro-carboxypeptidase Y that had been accumulated in the cytosol at the non-permissive temperature could be post-translationally translocated into the endoplasmic reticulum when cells were returned to the permissive temperature. This result indicates that post-translational translocation of preproteins across endoplasmic reticulum membranes can occur in vivo. We have also determined that the temperature-sensitive component is membrane-associated in ptl1, and that the membranes derived from this strain show a reversible temperature-sensitive translocation phenotype in vitro.     

Components of the Salmonella flagellar export apparatus and classification of export substrates

Until now, identification of components of the flagellar protein export apparatus has been indirect. We have now identified these components directly by establishing whether mutants defective in putative export components could translocate export substrates across the cytoplasmic membrane into the periplasmic space. Hook-type proteins could be exported to the periplasm of rod mutants, indicating that rod protein export does not have to precede hook-type protein export and therefore that both types of proteins belong to a single export class, the rod/hook-type class, which is distinct from the filament-type class. Hook-capping protein (FlgD) and hook protein (FlgE) required FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and FliR for their export to the periplasm. In the case of flagellin as an export substrate, because of the phenomenon of hook-to-filament switching of export specificity, it was necessary to use temperature-sensitive mutants and establish whether flagellin could be exported to the cell exterior following a shift from the permissive to the restrictive temperature. Again, FlhA, FlhB, FliH, FliI, and FliO were required for its export. No suitable temperature-sensitive fliQ or fliR mutants were available. FliP appeared not to be required for flagellin export, but we suspect that the temperature-sensitive FliP protein continued to function at the restrictive temperature if incorporated at the permissive temperature. Thus, we conclude that these eight proteins are general components of the flagellar export pathway. FliJ was necessary for export of hook-type proteins (FlgD and FlgE); we were unable to test whether FliJ is needed for export of filament-type proteins. We suspect that FliJ may be a cytoplasmic chaperone for the hook-type proteins and possibly also for FliE and the rod proteins. FlgJ was not required for the export of the hook-type proteins; again, because of lack of a suitable temperature-sensitive mutant, we were unable to test whether it was required for export of filament-type proteins. Finally, it was established that there is an interaction between the processes of outer ring assembly and of penetration of the outer membrane by the rod and nascent hook, the latter process being of course necessary for passage of export substrates into the external medium. During the brief transition stage from completion of rod assembly and initiation of hook assembly, the L ring and perhaps the capping protein FlgD can be regarded as bona fide export components, with the L ring being in a formal sense the equivalent of the outer membrane secretin structure of type III virulence factor export systems.     

Events in the endoplasmic reticulum abrogate the temperature sensitivity of Sindbis virus mutant ts23.

Temperature-sensitive mutations in proteins produced at or heated to a nonpermissive temperature render the proteins defective in some aspect of their maturation into functional entities. The characterization of temperature-sensitive mutations in model proteins, such as virus membrane proteins, has allowed the elucidation of critical events in the maturation of virus membranes as well as in the intracellular folding, processing, and transport of membrane proteins in general. We have used a transport-defective, temperature-sensitive mutant of Sindbis virus, ts23, which has two amino acid changes in the envelope protein E1, to further examine requirements placed upon the glycoproteins for their export to the plasma membrane. Pulse-chase experiments in which we utilized the transport inhibitors monensin and brefeldin A allowed us to synthesize and assemble the glycoproteins of ts23 into export-competent heterodimers at the permissive temperature while concurrently blocking their export to the cell surface. After removal of the inhibitors and a shift to the nonpermissive temperature, we assayed for protein transport, cell-cell fusion, and infectious-particle production. Taken together, the data show that the irreversible loss of the temperature-sensitive phenotype of ts23 can be correlated with the folding of E1 and the formation of export-competent PE2-E1 heterodimers in the endoplasmic reticulum. Furthermore, we have found that E1 pairs with PE2 to form the heterodimer prior to the completion of E1 folding.     

The E. coli divE mutation, which differentially inhibits synthesis of certain proteins, is in tRNASer1.

The temperature-sensitive divE mutant of Escherichia coli cannot synthesize certain membrane and cytoplasmic proteins at a non-permissive temperature. Growth of the mutant cells is arrested at a specific stage of the cell cycle when exposed to the non-permissive conditions, suggesting that the divE mutant possesses a defect in cell division control. From sequence determination of a cloned 1.35-kbp DNA fragment that complements the temperature-sensitive divE42 mutation, we characterized two genes in the segment ; one for tRNASer1 and the other for a 23 500 dalton protein. In parallel experiments we cloned the homologous 1.35-kbp DNA fragment from the divE42 mutant and determined its entire nucleotide sequence. Comparison of the two sequences showed that the mutation site is located not in the protein gene, but in the tRNA gene, where A10 is replaced by G10 in the D-stem. Lambda transducing phages carrying the subcloned tRNASer1 gene complemented the divE42 mutation, thereby confirming the conclusion obtained from sequence analyses of the fragments. This finding indicates that tRNASer1 is specifically involved in regulation of cell cycle-specific protein synthesis, coupled with an important step in the process of cell division, or that usage of serine tRNA is functionally specific for the biosynthesis of certain proteins.     

A role for unsaturated fatty acids in mitochondrial movement and inheritance

Yeast cells with the mdm2 mutation display temperature-sensitive growth and defective intracellular mitochondrial movement at the non-permissive temperature. The latter phenotype includes both an absence of mitochondrial transfer into daughter buds of mitotically growing cells and an aberrant mitochondrial distribution in cells exposed to mating pheromone. The wild-type MDM2 gene was cloned by complementation, and DNA sequence analysis revealed a large open reading frame encoding a putative protein of 58.4 kD. The predicted protein sequence is identical to that reported for the yeast OLE1 gene encoding fatty acid desaturase. Unsaturated fatty acid levels are substantially decreased in mdm2 cells after a prolonged incubation at the non-permissive temperature. The addition of oleic acid complements the temperature-sensitive growth and mitochondrial distribution defects of the mutant cells. These results indicate that mdm2 is a temperature-sensitive allele of OLE1 and demonstrate an essential role for unsaturated fatty acids in mitochondrial movement and inheritance.     

Characterization of a Temperature-Sensitive Vertebrate Clathrin Heavy Chain Mutant as a Tool to Study Clathrin-Dependent Events In Vivo

Clathrin and clathrin-dependent events are evolutionary conserved although it is believed that there are differences in the requirement for clathrin in yeast and higher vertebrates. Clathrin is a long-lived protein and thus, with clathrin knockdowns only long-term consequences of clathrin depletion can be studied. Here, we characterize the first vertebrate temperature-sensitive clathrin heavy chain mutant as a tool to investigate responses to rapid clathrin inactivation in higher eukaryotes. Although we created this mutant using a clathrin cryo-electron microscopy model and a yeast temperature-sensitive mutant as a guide, the resulting temperature-sensitive clathrin showed an altered phenotype compared to the corresponding yeast temperature-sensitive clathrin. First, it seemed to form stable triskelions at the non-permissive temperature although endocytosis was impaired under these conditions. Secondly, as a likely consequence of the stable triskelions at the non-permissive temperature, clathrin also localized correctly to its target membranes. Thirdly, we did not observe missorting of the lysosomal enzyme beta-glucuronidase which could indicate that the temperature-sensitive clathrin is still operating at the non-permissive temperature at the Golgi or, that, like in yeast, more than one TGN trafficking pathway exists. Fourthly, in contrast to yeast, actin does not appear to actively compensate in general endocytosis. Thus, there seem to be differences between vertebrates and yeast which can be studied in further detail with this newly created tool.     

Isolation and analysis of a mammalian temperature-sensitive mutant defective in G2 functions

A temperature-sensitive (ts) mutant, designated tsFT210, was isolated from a mouse mammary carcinoma cell line, FM3A. The tsFT210 cells grew normally at 33 degrees C (permissive temperature), but more than 80% of the cells were arrested at the G2 phase at 39 degrees C (non-permissive temperature) as revealed by flow-microfluorimetric analysis. DNA replication and synthesis of other macromolecules by this mutant seemed to be normal at 39 degrees C for at least 10 h. However, in this mutant, hyperphosphorylation of H1 histone from the G2 to M phase, which occurs in the normal cell cycle, could not be detected at the non-permissive temperature. This suggests that a gene product which is temperature-sensitive in tsFT210 cells is necessary for hyperphosphorylation of H1 histone and that this gene product may be related to chromosome condensation.     

Isolation and characterization of novel mutations in CDC50, the non-catalytic subunit of the Drs2p phospholipid flippase

Flippases (type 4 P-type ATPases) are believed to translocate phospholipids from the exoplasmic to the cytoplasmic leaflet in bilayer membranes. Since flippases are structurally similar to ion-transporting P-type ATPases such as the Ca(2+) ATPase, one important question is how flippases have evolved to transport phospholipids instead of ions. We previously showed that a conserved membrane protein, Cdc50p, is required for the endoplasmic reticulum exit of the Drs2p flippase in yeast. However, Cdc50p is still associated with Drs2p after its transport to the endosomal/trans-Golgi network (TGN) membranes, and its function in the complex with Drs2p is unknown. In this study, we isolated novel temperature-sensitive (ts) cdc50 mutants whose products were still localized to endosomal/TGN compartments at the non-permissive temperature. Mutant Cdc50 proteins colocalized with Drs2p in endosomal/TGN compartments, and they co-immunoprecipitated with Drs2p. These cdc50-ts mutants exhibited defects in vesicle transport from early endosomes to the TGN as the cdc50 deletion mutant did. These results suggest that mutant Cdc50 proteins could be complexed with Drs2p, but the resulting Cdc50p-Drs2p complex is functionally defective at the non-permissive temperature. Cdc50p may play an important role for phospholipid translocation by Drs2p.     

Effects of secA mutations on the synthesis and secretion of proteins in Escherichia coli. Evidence for a major export system for cell envelope proteins

We have followed the synthesis and secretion of a number of periplasmic and outer membrane proteins in three strains of Escherichia coli, a secA amber mutant, a secA temperature-sensitive mutant, and a strain that blocks protein secretion due to a high level of expression of an export-defective hybrid protein between maltose-binding protein and beta-galactosidase (MalE-LacZ). Our results show that after several hours under nonpermissive conditions the specificity and extent of the export blocks in the secA temperature-sensitive mutant and the strain producing the MalE-LacZ hybrid protein are identical, affecting at least four major outer membrane proteins and most but not all periplasmic proteins. The secA gene product, therefore, appears to be an essential component of the major export pathway in E. coli which is used by many envelope proteins independent of whether they are cotranslationally or post-translationally secreted. In contrast, the synthesis of only a subset of these envelope proteins is reduced in the secA amber mutant after shift to the nonpermissive condition. These results indicate that the SecA protein serves roles both in the synthesis and the secretion of certain cell envelope proteins.     

Isolation of temperature-sensitive mutants from murine leukemic cells (L5178Y)

Two temperature-sensitive mutants (ts1 and ts3) have been isolated from murine leukemic cells, L5178Y, after mutagenesis and cytosine arabinoside selection. Both ts1 and ts3 grew normally at the permissive temperature (33 °C) but not at the non-permissive temperature (39 °C). Consistent results were obtained with the growth patterns in suspension culture as well as the plating efficiencies in soft agar. Temperature shift experiments showed that the mutant cells remained viable after extended exposure to the non-permissive temperature. Labeling studies with radioactive precursors indicated that the synthesis of DNA, but not of RNA or protein, was affected in these mutants at 39 °C. The defective function of ts3 cells was substantially corrected by supplementing alanine, hypoxanthine, and pyruvate.     

SCD1 is required for cytokinesis and polarized cell expansion in Arabidopsis thaliana [corrected

In the leaf epidermis, guard mother cells undergo a stereotyped symmetric division to form the guard cells of stomata. We have identified a temperature-sensitive Arabidopsis mutant, stomatal cytokinesis-defective 1-1 (scd1-1), which affects this specialized division. At the non-permissive temperature, 22 degrees C, defective scd1-1 guard cells are binucleate, and the formation of their ventral cell walls is incomplete. Cytokinesis was also disrupted in other types of epidermal cells such as pavement cells. Further phenotypic analysis of scd1-1 indicated a role for SCD1 in seedling growth, root elongation and flower morphogenesis. More severe scd1 T-DNA insertion alleles (scd1-2 and scd1-3) markedly affect polar cell expansion, most notably in trichomes and root hairs. SCD1 is a unique gene in Arabidopsis that encodes a protein related to animal proteins that regulate intracellular protein transport and/or mitogen-activated protein kinase signaling pathways. Consistent with a role for SCD1 in membrane trafficking, secretory vesicles were found to accumulate in cytokinesis-defective scd1 cells. In addition the scd1 mutant phenotype was enhanced by low doses of inhibitors of cell plate consolidation and vesicle secretion. We propose that SCD1 functions in polarized vesicle trafficking during plant cytokinesis and cell expansion.     

Genetic control of DNA initiation in Escherichia coli

We describe the isolation, and properties of a mutant (CT28) of Escherichia coli with a temperature-sensitive defect in DNA initiation that is reversible. The mutation (dna-28) responsible for this defect is shown to be located in the same region of the map as the dnaC group of DNA initiation mutants.A terminalized culture of CT28 initiates DNA synthesis synchronously immediately upon lowering the temperature, and will do so in the presence of chloram-phenicol.During prolonged incubation at the non-permissive temperature, the cells accumulate a capacity to initiate multiple rounds of replication per chromosome.The variation in the susceptibility of the argH^? and thyA^? alleles to reversion by pulse mutagenesis with nitrosoguanidine during a synchronous round of DNA replication, suggests that this round of replication is bidirectional and commences from an origin in the vicinity of 60 to 65 minutes.CT28 contains two temperature-sensitive mutations. These have been mapped and separated into two derivative strains. One of these, CT28-3b, carries the dna-28 mutation of the C locus, and like the parental double mutant is reversibly temperature-sensitive for an initiation function; but it is more temperature-sensitive than either the double mutant or the other single mutant derivative, CT28-1. The other, CT28-1, is not defective in DNA replication or initiation of replication at the non-permissive temperature.     

Functional analysis of the signal recognition particle in Escherichia coli by characterization of a temperature-sensitive ffh mutant

The Ffh protein of Escherichia coli is a 48-kDa polypeptide that is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP). Efforts to understand the function of Ffh in bacteria have depended largely on the use of E. coli strains that allow depletion of the wild-type gene product. As an alternative approach to studying Ffh, a temperature-sensitive ffh mutant was isolated. The ffh-10(Ts) mutation results in two amino acid changes in conserved regions of the Ffh protein, and characterization of the mutant revealed that the cells rapidly lose viability at the nonpermissive temperature of 42 degrees C as well as show reduced growth at the permissive temperature of 30 degrees C. While the ffh mutant is defective in insertion of inner membrane proteins, the export of proteins with cleavable signal sequences is not impaired. The mutant also shows elevated expression of heat shock proteins and accumulates insoluble proteins, especially at 42 degrees C. It was further observed that the temperature sensitivity of the ffh mutant was suppressed by overproduction of 4.5S RNA, the RNA component of the bacterial SRP, by stabilizing the thermolabile protein. Collectively, these results are consistent with a model in which Ffh is required only for localization of proteins integral to the cytoplasmic membrane and suggest new genetic approaches to the study of how the structure of the SRP contributes to its function.     

Mutants of Bacillus subtilis affected in spore outgrowth.

Six mutants of Bacillus subtilis 168 that are temperature-sensitive in spore outgrowth were isolated. The outgrowth process proceeds normally at 35 degrees C, but at the non-permissive temperature (47 degrees C) it is arrested at a specific stage characteristic for each mutant strain. The mutants are not altered in vegetative growth whether at 35 degrees C or at 47 degrees C. They were characterized for their ability to synthesize RNA, proteins and DNA during outgrowth. A mutant defective in spore germination was also isolated; less than 5% of its spores can germinate at any of the temperatures tested. The mutations were mapped by means of transduction and transformation. The isolation of a number of outgrowth mutants which map at different loci and which affect outgrowth at different times is discussed in relation to the regulation of this process.     

Genetic identification of an endoproteinase encoded by the adenovirus genome

The temperature-sensitive adenovirus type 2 mutant H2ts1 is defective for polypeptide processing at the non-permissive temperature. We have in the present study mapped the mutation by marker rescue and DNA sequencing techniques: the mutation is a C/T transition located at map co-ordinate 61.1. Previous sequencing studies have identified an uninterrupted translational reading frame in this part of the adenovirus genome, encoding a hypothetical 23 X 10(3) Mr polypeptide. The mutation leads to a proline/leucine substitution in the 23 X 10(3) Mr polypeptide.