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K R Willison

Publications and source records attributed to K R Willison.

At least 37 records · Page 2Linked to original sources
PubMed1997

Elucidation of the subunit orientation in CCT (chaperonin containing TCP1) from the subunit composition of CCT micro-complexes.

A collection of chaperonin containing TCP1 (CCT) micro-complexes that are comprised of subsets of the constitutively expressed CCT subunits have been identified. These CCT micro-complexes have mol. wts ranging from 120 to 250 kDa and are present in cells at lower abundance (<5%) as compared with intact CCT. Biochemical characterization of these microcomplexes has shown that several are comprised of two different types of CCT subunit. Furthermore, it was observed that each subunit associates with only one or two other different types of subunit, suggesting that each subunit has fixed partners. This observation, together with CCT gene counting being concordant with the 8-fold structural symmetry, is consistent with predictions derived from analysis of the primary structures of these subunits concerning inter-subunit interactions, and implies a unique topology of the subunits constituting the torodial ring in CCT. The series of subunit-subunit association patterns determined from CCT micro-complexes has provided information to infer, from the 5040 (7!factorial) combinatorial possibilities, one probable subunit orientation within the torodial ring.

Animals↗
PubMed1997

Tissue-specific subunit of the mouse cytosolic chaperonin-containing TCP-1.

We have cloned a novel Tcp-1-related mouse testis cDNA encoding a polypeptide of 531 amino acids which shares 81.2% identity with the zeta subunit of the mouse cytosolic chaperonin-containing TCP-1 (CCT). Immunoblot analysis of mouse testis CCT subunits separated by 2-dimensional gel electrophoresis indicates that this novel gene, Cctz-2, encodes a CCT subunit of Mr 57 000 and pI 7.1. Cctz-2 mRNA is detected only in testis whereas the other Cctz gene, Cctz-1, is expressed in all tissues investigated. The CCTzeta-2 subunit may have specific functions in the folding of testicular proteins and for interactions with testicular molecular chaperones.

Amino Acid Sequence↗
PubMed1996

ADP-ribosylation factor 1-regulated phospholipase D activity is localized at the plasma membrane and intracellular organelles in HL60 cells.

ADP-ribosylation factor (ARF), a small GTPase required for vesicle formation, has been identified as an activator of phospholipase D (PLD), thus implying that PLD is localized at intracellular organelles. HL60 cells were prelabelled with [14C]acetate for 72 h and, after disruption, fractionated on a linear sucrose gradient. ARF1-regulated PLD activity in each fraction was assessed by measurement of phosphatidylethanol production. Two peaks of activity were identified, coincident with markers for Golgi/endoplasmic reticulum/granules (endomembranes) and plasma membrane respectively. Analysis of the fractions using exogenous phosphatidylcholine as substrate confirmed the presence of ARF1-dependent PLD activity in endomembranes and plasma membrane, and also identified an additional activity in the cytosol. In formyl-Met-Leu-Phe-stimulated cells, PLD activity as assessed by phosphatidylethanol formation was also associated with both the plasma membrane and endomembranes. Since ARF1-regulated PLD activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), the distributions of inositol lipids and the kinases responsible for lipid phosphorylation were examined. PIP2 was highly enriched at the plasma membrane, whereas phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PI4P), the precursors for PIP2 synthesis, were found predominantly at endomembranes. The distribution of PI 4-kinase and PI4P 5-kinase activities confirmed the plasma membrane as the major site of PIP2 production. However, endomembranes possessed substantial PI 4-kinase activity and some PI4P 5-kinase activity, illustrating the potential for PIP2 synthesis. It is concluded that:(1) ARF1-regulated PLD activity is localized at endomembranes and the plasma membrane, (2) PIP2 is available at both membrane compartments to function as a cofactor for ARF-regulated PLD, and (3) in intact cells, formyl-Met-Leu-Phe stimulates PLD activity at endomembranes as well as plasma membrane.

1-Phosphatidylinositol 4-Kinase↗
PubMed1996

Analysis of chaperonin-containing TCP-1 subunits in the human keratinocyte two-dimensional protein database: further characterisation of antibodies to individual subunits.

The chaperonin-containing TCP-1 (CCT), found in the eukaryotic cytosol, is currently the focus of extensive research. CCT consists of at least eight different subunit types encoded by independent but related genes, and a set of antibodies that recognise individual subunits has proved useful in the characterisation and functional analysis of CCT. These antibodies were used to identify subunits of CCT in the human keratinocyte two-dimensional protein database. Accurate values for the pI and molecular mass of human CCT subunits were determined from the database, and biological data was obtained regarding changes in subunit levels in response to extracellular agents and growth conditions. The second part of the study describes the characterisation of seven monoclonal antibodies raised against mouse TCP-1, also known as CCT alpha, using a combination of epitope mapping and immunoblot analysis of protein extracts from different species and tissue types. Some antibodies were not monospecific for TCP-1, and a number of epitope-related proteins were identified.

Animals↗
PubMed1996

Peptide mass fingerprinting of chaperonin-containing TCP-1 (CCT) and copurifying proteins.

The chaperonin-containing TCP-1 (CCT), found in the eukaryotic cytosol, is currently the focus of extensive research, CCT isolated from mouse testis lysate sediments at 20S in a sucrose gradient and accounts for about 70% of the total protein in this fraction. We intend to identify all the other proteins that copurify with CCT and to compile a reference profile for future studies. Their identification can be accelerated by a combination of protease digestion, matrix-assisted laser desorption-mass spectrometry, and database matching known as peptide mass fingerprinting. We applied this strategy to 32 polypeptides resolved by 2-dimensional gel electrophoresis, and 23 known proteins and 6 novel proteins were identified. We analyzed isoelectric variants of the CCT subunits and differences in the peptide mass spectra of two CCT theta isoforms indicated a novel posttranslational modification of this subunit.

Amino Acid Sequence↗
PubMed1996

Brefeldin A and mannose 6-phosphate regulation of acrosomic related vesicular trafficking.

Acrosomal biogenesis represents a unique system for the molecular analysis of the various processes involved in vesicular membrane transport. To assess the basic membrane trafficking mechanisms used in spermatids, we have used two fluorescent lipid compounds that label: a) the Golgi and Golgi-derived vesicles (C5-DMB-Cer), and b) endocytic vesicles (FM4-64). Incubation of mouse testicular germ cells at 33 degrees C for 1.5 h with C5-DMB-Cer revealed that C5-DMB-Cer labeling is localized in the Golgi and acrosome of early-maid round spermatid stages, with no labeling of the acrosome in late round spermatid stages. Culturing germ cells with FM4-64 for 1.5 h at 33 degrees C, showed that FM4-64 labeling in spermatids was localized in endocytic vesicles and Golgi of early-mid round spermatid stages, whereas in a small population of late round spermatid stages, FM4-64 was also localized in the apex region of the acrosome. Incubation with brefeldin A (BFA) (5 micrograms/ml) inhibited the distribution of C5-DMB-Cer and FM4-64 to the acrosome, however, it did not affect the localization of acrosin-an acrosome-specific protein-indicating that there was no apparent acrosome disassembly in the presence of BFA. Localization of FM4-64 in late round spermatids in the presence of 2.5 mM mannose 6-phosphate was found in endocytic vesicles and the Golgi, but not the acrosome. These results show that there are at least two sources of vesicular transport to the acrosome derived from the Golgi and plasma membrane.

Acrosin↗
PubMed1995

Antibody characterisation of two distinct conformations of the chaperonin-containing TCP-1 from mouse testis.

We describe a panel of antibodies specific to individual subunits of the chaperonin-containing TCP-1 (CCT) and one antibody that reacts with all the subunits of CCT. Immunoblot analysis of CCT purified from mouse testis suggests that the testis-specific subunit, S6, may be related to CCT zeta and that a co-purifying 63 kDa protein may be a novel subunit of CCT. Using these antibodies in the analysis of CCT subjected to nondenaturing IEF we observed the resolution of two distinct conformations of CCT, which differ in their susceptibility to proteolysis and in the number of associated polypeptides.

Animals↗
PubMed1994

Cystosolic chaperonin subunits have a conserved ATPase domain but diverged polypeptide-binding domains.

CCT (also called the TCP-1 complex or TriC) is a chaperonin found in the eukaryotic cytosol, and has unique structural and functional features. Unlike homo-oligomeric chaperonins, CCT comprises at least eight different subunits, and appears to have a limited range of physiological substrates. We have analysed CCT sequences in light of the recent determination of the crystal structure and mutational identification of the functional domains of the bacterial chaperonin GroEL. A high level of identity among all chaperonin subunits is observed in those regions that correspond to the ATP-binding site of GroEL. By contrast, no significant identity is shared in the region corresponding to the polypeptide-binding region of GroEL, either between CCT subunits or between CCT subunits and GroEL. This suggests that the polypeptide-binding sites of CCT subunits have diverged both from each other and from GroEL, which may explain the apparently different range of substrates recognized by CCT.

Adenosine Triphosphatases↗
PubMed1993

Protein folding in the cell: functions of two families of molecular chaperone, hsp 60 and TF55-TCP1.

Two families of molecular chaperone, the hsp 60-GroEL family and the TF55-TCP1 family, have been discovered in evolutionarily related cellular compartments. A member of one of these families, hsp 60, has been shown to play a global role in polypeptide chain folding in mitochondria. We review here studies of both hsp 60 and other family members, discussing their essential physiological roles and mechanism of action.

Animals↗
PubMed1993

Cloning, chromosomal localization and expression pattern of the POU domain gene Oct-11.

POU domain genes encode a family of highly conserved transacting factors that influence the transcriptional activity of several cell type-specific and ubiquitous genes. We have cloned and sequenced cDNAs encoding a novel mouse POU domain protein, Oct-11, that is closely related within the POU domain to the POU class II proteins, Oct-1 and Oct-2. Recombinant Oct-11 protein binds specifically to an octamer sequence in vitro. The Oct-11 gene is expressed during mouse embryogenesis and in the adult thymus and testis. In addition, it is abundant in the myeloma cell line P3/NS-1/1-Ag4.1. We describe the structure of Oct-11 and its chromosomal localization, and discuss the evidence that the POU class II gene family has evolved by duplication and divergence of a common ancestral gene.

Amino Acid Sequence↗
PubMed1993

A 102 kDa subunit of a Golgi-associated particle has homology to beta subunits of trimeric G proteins.

We have identified a 102 kDa protein, p102, which is found on the cytoplasmic face of Golgi membranes, exocytic transport vesicles and in the cytosol. A monoclonal antibody that cross-reacts with p102 is able to immunoprecipitate a 500-600 kDa protein complex containing p102 and additional subunits. The composition of this p102-containing protein complex resembles that of the Golgi coatomer complex, which constitutes the coat of non-clathrin coated vesicles. One of the subunits of the p102 complex reacts with a monoclonal antibody that detects beta-COP, a subunit of the Golgi coatomer complex. Like beta-COP, p102 exists in a brefeldin A-sensitive association with Golgi membranes. The sequence of p102 contains an N-terminal domain composed of six repeats which are similar to those found in the beta subunit of trimeric G proteins and other regulatory proteins. We suggest that p102 may be involved in regulating membrane traffic in the constitutive exocytic pathway.

Amino Acid Sequence↗
PubMed1991

Sequence of the t complex Tcp-10at gene and examination of the Tcp-10t gene family.

Transmission ratio distortion (TRD) is a property of complete t haplotypes which results in the preferential transmission of the t haplotype chromosome from heterozygous t/+ males to the majority of the offspring. A candidate gene for one of the primary genetic elements in TRD, the t complex responder locus has recently been suggested to be Tcp-10bt. There are multiple, functional Tcp-10t genes, but genetic data suggest the presence of the Tcp-10at gene alone is compatible with normal transmission ratios. Here we present the complete sequence and genomic structure of the Tcp-10at gene which is compared with sequence data from a number of cDNAs and genomic subclones representing all active Tcp-10t family genes. A detailed table of all sequence variants discovered in the course of our investigation is presented, and we have clarified the extent of 5' untranslated alternative splicing patterns exhibited by this gene family. A 60 base pair (bp) in-frame deletion from the 5' end of exon 3 of the Tcp-10at gene is also presented and compared with the equivalent region of Tcp-10bt and Tcp-10ct. A search of the University of Edinburgh database has revealed a significant homology between the Tcp-10bt open reading frame and several cytosolic filament proteins. Interestingly, the region of homology is involved in the deletion from the Tcp-10at gene.

Alleles↗
PubMed1989

Chromosomal localization of zinc finger protein genes in man and mouse.

We have determined the mouse and human chromosomal location of a gene (Zfp-3) that codes for a protein that contains potential DNA zinc-binding fingers. An analysis of the segregation of restriction fragment length polymorphisms in recombinant inbred strains and in an interspecific backcross demonstrated that Zfp-3 is located on mouse chromosome 11. Zfp-3 is very closely linked to the Trp53-1 locus but unlinked to another finger protein gene Zfp-4 located on mouse chromosome 8. In humans ZFP3 has been localized to chromosome 17p12-17pter and thus is part of the conserved linkage group between this chromosome and the distal half of mouse chromosome 11.

Animals↗
PubMed1988

Extent of the mouse t complex and its inversions shown by in situ hybridization.

Probes for loci situated near one end of the proximal (Tcp-1) and distal (Qa-2, 3) inversions of the mouse t complex have been hybridized to chromosomes of mice with and without t complexes and with morphologically distinguishable chromosome 17s. Both the probe for Tcp-1 and that for Qa-2, 3 hybridized to clearly different positions on t and non-t chromosomes, thus making visible the extent of the two inversions. The proximal inversion extends from roughly the junction of bands A1 and A2 to band A3, and the distal inversion from band A3 to band C. Thus, the whole t complex extends from the band A1-A2 junction to band C, and is therefore somewhat larger than previously thought, and occupies about 1.2% of the genome. A probe for complement component 3 (C3-1), genetically known to be several cM distal to the t complex, was found by in situ hybridization to lie in band E1. The proximal part of chromosome 17 is one of the best known parts of the mouse genome, at both the genetic and molecular levels. It may soon be possible to correlate the length of the t complex in terms of chromosomal distance with its physical length in megabases.

Animals↗
PubMed1986

Molecular cloning and sequence analysis of a haploid expressed gene encoding t complex polypeptide 1.

The mouse t haplotypes show defects in spermatogenesis attributed to multiple loci on chromosome 17. We have cloned the gene for an abundant testicular germ cell protein, t complex polypeptide 1, which has a variant form in t haplotypes, TCP-1A. A cDNA clone, pB1.4, which hybridizes to a 19S mRNA that is abundant in haploid cells during mouse spermatogenesis, derives from the 3' end of the mRNA encoding TCP-1B. The Tcp-1 gene appears to be a member of a novel gene family and shows multiple changes between the predicted amino acid sequences of TCP-1B and TCP-1A. An additional Taq1 site is created by a T to C transition in the predicted open reading frame of the Tcp-1a gene. The resultant RFLP has allowed typing of the Tcp-1 gene cluster in 54 complete and partial t haplotype chromosomes. DNA sequence comparison of the Tcp-1 genes suggests that the t haplotype chromosome arose within the genus Mus more than one million years ago.

Animals↗