Biomedical subjects

D McCollum

Publications and source records attributed to D McCollum.

27 records · Page 2Linked to original sources

PubMed1996

Fission yeast Sop2p: a novel and evolutionarily conserved protein that interacts with Arp3p and modulates profilin function.

Profilins bind to monomeric actin and also interact with ligands such as phosphoinositide 4,5-bisphosphate, the proline-rich protein VASP and a complex of four to six polypeptides identified in Acanthamoeba that includes two actin-related proteins. Here, we report the identification and characterization of an essential gene from Schizosaccharomyces pombe, sop2+, a mutation in which rescues the temperature-sensitive lethality of a profilin mutation, cdc3-124. The sop2-1 mutant is defective for cell elongation and septation, suggesting that it is involved in multiple cortical actin-requiring processes. Consistent with a role in actin cytoskeletal function, negative interactions have been identified between sop2-1 and act1-48, a mutant allele of actin. Sop2p is a novel 377 amino acid polypeptide with similarity to proteins of the beta-transducin repeat family. Sop2p-related proteins have been identified by sequencing projects in diverse species, and we have isolated a human cDNA highly related to sop2+, SOP2 Hs, which functionally complements the sop2-1 mutation. Sop2p proteins from all species contain peptide sequences identical or highly similar to two peptide sequences from an Acanthamoeba beta-transducin repeat protein present in the profilin binding complex. Biochemical analyses demonstrate that Sop2p is present in a complex which also contains the actin-related protein, Arp3p. Immunofluorescence studies reveal the presence of Sop2p in (i) punctate structures distributed throughout the cell, (ii) cables that extend the length of the cell, and (iii) a medial band in a small percentage of septating cells. Collectively these data demonstrate the interaction of Sop2p with Arp3p, profilin and actin.

Acanthamoeba↗

PubMed1996

The Schizosaccharomyces pombe actin-related protein, Arp3, is a component of the cortical actin cytoskeleton and interacts with profilin.

The gene encoding the actin-related protein Arp3 was first identified in the fission yeast Schizosaccharomyces pombe and is a member of an evolutionarily conserved family of actin-related proteins. Here we present several key findings that define an essential role for Arp3p in the functioning of the cortical actin cytoskeleton. First, mutants in arp3 interact specifically with profilin and actin mutants. Second, Arp3 localizes to cortical actin patches which are required for polarized cell growth. Third, the arp3 gene is required for the reorganization of the actin cytoskeleton during the cell cycle. Finally, the Arp3 protein is present in a large protein complex. We believe that this complex may mediate the cortical functions of profilin at actin patches in S. pombe.

Actin-Related Protein 3↗

PubMed1995

The Pichia pastoris peroxisomal protein PAS8p is the receptor for the C-terminal tripeptide peroxisomal targeting signal.

The peroxisomal targeting signal 1 (PTS1), consisting of a C-terminal tripeptide (SKL and variants), directs polypeptides to the peroxisome matrix in evolutionarily diverse organisms. Previous studies in the methylotrophic yeast Pichia pastoris identified a 68 kDa protein, PAS8p, as a potential component of the PTS1 import machinery. We now report several new properties of this molecule which, taken together, show that it is the peroxisomal PTS1 receptor. (i) PAS8p is localized to and tightly associated with the cytoplasmic side of the peroxisomal membrane, (ii) peroxisomes of wild-type, but not of pas8 delta (null) mutant, P.pastoris cells bind a PTS1-containing peptide (CRYHLKPLQSKL), (iii) CRYHLKPLQSKL can be cross-linked to PAS8p after binding at the peroxisome membrane and (iv) purified PAS8p binds CRYHLKPLQSKL with high affinity (nanomolar dissociation constant). In addition, the tetratricopeptide repeat (TPR) domain of PAS8p is identified as the PTS1 binding region.

Amino Acid Sequence↗

PubMed1995

Schizosaccharomyces pombe cdc4+ gene encodes a novel EF-hand protein essential for cytokinesis.

Schizosaccharomyces pombe cells divide by medial fission. One class of cell division mutants (cdc), the late septation mutants, defines four genes: cdc3, cdc4, cdc8, and cdc12 (Nurse, P., P. Thuriaux, and K. Nasmyth. 1976. Mol. & Gen. Genet. 146:167-178). We have cloned and characterized the cdc4 gene and show that the predicted gene product. Cdc4p, is a 141-amino acid polypeptide that is similar in sequence to EF-hand proteins including myosin light chains, calmodulin, and troponin C. Two temperature-sensitive lethal alleles, cdc4-8 and cdc4-31, accumulate multiple nuclei and multiple improper F-actin rings and septa but fail to complete cytokinesis. Deletion of cdc4 also results in a lethal terminal phenotype characterized by multinucleate, elongated cells that fail to complete cytokinesis. Sequence comparisons suggest that Cdc4p may be a member of a new class of EF-hand proteins. Cdc4p localizes to a ringlike structure in the medial region of cells undergoing cytokinesis. Thus, Cdc4p appears to be an essential component of the F-actin contractile ring. We find that Cdc4 protein forms a complex with a 200-kD protein which can be cross-linked to UTP, a property common to myosin heavy chains. Together these results suggest that Cdc4p may be a novel myosin light chain.

Amino Acid Sequence↗

PubMed1994

The Schizosaccharomyces pombe cdc5+ gene encodes an essential protein with homology to c-Myb.

The Schizosaccharomyces pombe cdc5+ gene was identified in the first screen for cell division cycle mutants in this yeast. The cdc5+ gene was reported to be required for nuclear division but because of its modest elongation and leaky nature at the non-permissive temperature, it was not investigated further. Here, we report the characterization of the single allele of this gene, cdc5-120, in more detail. The mutant arrests with a 2N DNA content and a single interphase nucleus. Further genetic analyses suggest that cdc5+ gene function is essential in the G2 phase of the cell cycle. We have cloned and sequenced the cdc5+ gene. The deduced protein sequence predicts that Cdc5 is an 87 kDa protein and contains a region sharing significant homology with the DNA binding domain of the Myb family of transcription factors. Deletion mapping of the cdc5+ gene has shown that the N-terminal 232 amino acids of the protein, which contain the Myb-related region, are sufficient to complement the cdc5ts strain. A cdc5 null mutant was generated by homologous recombination. Haploid cells lacking cdc5+ are inviable, indicating that cdc5+ is an essential gene. A fusion protein consisting of bacterial glutathione S-transferase joined in-frame to the N-terminal 127 amino acids of the Cdc5 protein is able to bind to DNA cellulose at low salt concentrations. This evidence suggests that cdc5+ might encode a transcription factor whose activity is required for cell cycle progression and growth during G2.

Amino Acid Sequence↗

PubMed1994

Surgery for angina pectoris: a historical perspective.

Coronary artery disease continues to be the major cause of death in the United States each year with close to 1 million people dying of this disease annually, one third of those being under the age of 65. An estimated 5 million people in this country suffer some degree of disability from angina pectoris.

Angina Pectoris↗

PubMed1993

The pas8 mutant of Pichia pastoris exhibits the peroxisomal protein import deficiencies of Zellweger syndrome cells--the PAS8 protein binds to the COOH-terminal tripeptide peroxisomal targeting signal, and is a member of the TPR protein family.

We previously described the isolation of mutants of the yeast Pichia pastoris that are deficient in peroxisome assembly (pas mutants). We describe the characterization of one of these mutants, pas8, and the cloning of the PAS8 gene. The pas8 mutant is deficient for growth, but not for division or segregation of peroxisomes, or for induction of peroxisomal proteins. Two distinct peroxisomal targeting signals, PTS1 and PTS2, have been identified that are sufficient to direct proteins to the peroxisomal matrix. We show that the pas8 mutant is deficient in the import of proteins with the PTS1, but not the PTS2, targeting signal. This is the same import deficiency as that found in cells from patients with the lethal human peroxisomal disorder Zellweger syndrome. Cloning and sequencing of the PAS8 gene reveals that it is a novel member of the tetratricopeptide repeat gene family. Antibodies raised against bacterially expressed PAS8 are used to show that PAS8 is a peroxisomal, membrane-associated protein. Also, we have found that in vitro translated PAS8 protein is capable of binding the PTS1 targeting signal specifically, raising the possibility that PAS8 is a PTS1 receptor.

Acetyl-CoA C-Acetyltransferase↗

PubMed1992

Development of the yeast Pichia pastoris as a model organism for a genetic and molecular analysis of peroxisome assembly.

We describe the isolation of mutants of the yeast Pichia pastoris that are deficient in peroxisome assembly (pas). These mutants of P. pastoris can be identified solely by their inability to grow on methanol and oleic acid, the utilization of which requires peroxisomal enzymes, and are defined by the absence of normal peroxisomes as judged by electron microscopy and biochemical fractionation experiments. These mutants are the result of genetic defects at single loci and represent at least eight different complementation groups. The isolation of pas mutants of P. pastoris by a simple screen for mutants unable to use methanol and oleic acid represents a significantly more efficient method for identification of pas mutants than is possible in other organisms. To exploit this advantage fully we also developed new reagents for the genetic and molecular manipulation of P. pastoris. These include a set of auxotrophic strains with an essentially wild-type genetic background, plasmids that act as Escherichia coli-P. pastoris shuttle vectors, and genomic DNA libraries for isolation of P. pastoris genes by functional complementation of mutants or by nucleic acid hybridization. The availability of numerous pas mutants and the reagents necessary for their molecular analysis should lead to the isolation and characterization of genes involved in peroxisome assembly.

Biological Transport↗

PubMed1992

An efficient screen for peroxisome-deficient mutants of Pichia pastoris.

We describe a rapid and efficient screen for peroxisome-deficient (per) mutants in the yeast Pichia pastoris. The screen relies on the unusual ability of P. pastoris to grow on two carbon sources, methanol and oleic acid, both of which absolutely require peroxisomes to be metabolized. A collection of 280 methanol utilization-defective (Mut-) P. pastoris mutants was isolated, organized into 46 complementation groups, and tested for those that were also oleate-utilization defective (Out-) but still capable of growth on ethanol and glucose. Mutants in 10 groups met this phenotypic description, and 8 of these were observed by electron microscopy to be peroxisome deficient (Per-). In each per mutant, Mut-, Out-, and Per- phenotypes were tightly linked and therefore were most likely due to a mutation at a single locus. Subcellular fractionation experiments indicated that the peroxisomal marker enzyme catalase was mislocalized to the cytosol in both methanol- and oleate-induced cultures of the mutants. In contrast, alcohol oxidase, a peroxisomal methanol utilization pathway enzyme, was virtually absent from per mutant cells. The relative ease of per mutant isolation in P. pastoris, in conjunction with well-developed procedures for its molecular and genetic manipulation, makes this organism an attractive system for studies on peroxisome biogenesis.

Acyl-CoA Oxidase↗