Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 92-98).

Includes bibliographical references (p. 64-71).

published_or_final_version / Zoology / Master / Master of Philosophy

Oxysterol binding protein (OSBP) binds 25-hydroxycholesterol (25OH) and cholesterol, which regulates PH and FFAT domain interaction with the Golgi apparatus and endoplasmic reticulum, respectively. Adjacent to these domains is a phosphorylated serine-rich motif (SRM, T379, S381, S384, S387, S388, S391) that we hypothesize controls sterol transport by OSBP. To test this, OSBP dephospho-mimics or phospho-mimics were expressed in CHO cells. Western blot analysis showed that the S381 is phosphorylated by PKA and is required for phosphorylation of down-stream serine residues. When expressed in OSBP-null CHO cells, there was no difference in the localization of the OSBP mutants, and all mutants restored SM synthesis in response to 25OH. Recombinant OSBP 5S?5E had increased cholesterol binding and extraction, and decreased cholesterol transfer to liposomes compared to OSBP. OSBP 5S?5E also bound VAP more efficiently. A model is proposed wherein SRM phosphorylation facilitates VAP association with the ER and increases cholesterol extraction.

Using a novel purification scheme and a new assay for detection of DNA kinase activity, a Polymin P-precipitable DNA kinase has been identified and characterized from calf thymus extracts. The DNA kinase activity was able to phosphorylate RNA as well as single-stranded and double-stranded DNA, therefore it has been termed Polymin P-precipitable polynucleotide kinase (PP-PNK). The enzyme had a neutral to alkaline, broad pH optimum that distinguished it from the previously described mammalian DNA kinases that have an acidic pH optimum. The sedimentation coefficient of the enzyme was 3.4-3.8 S, indicating a molecular weight of about 50 kDa. Estimates for the K$ sb{ rm M}$ for ATP were 52 $ mu$M and for the oligonucleotide substrate 8 $ mu$M. The activity was inhibited by pyrophosphate anions and to a lesser extent by sulfate anions. These results differentiate PP-PNK from other mammalian polynucleotide kinases.

Whole cell extracts from fresh calf thymus glands were subjected to Polymin P fractionation and Q Sepharose chromatography. Three peaks of DNA kinase activity, designated SNQI, SNQII and SNQIII, were found in the supernatant fraction. Studies of SNQI have revealed an estimated molecular mass of 50-90 kDa by Superose 12 chromatography, and activity gel analysis following SDS-PAGE identified an active polypeptide of approximately 55 kDa. This enzyme preparation, purified 10,000-fold, phosphorylated 5$ sp prime$-OH-terminated oligodeoxyribonucleotides and double stranded DNA, yet was inactive on an oligoriboadenosine ladder. SNQI functions optimally at an acidic pH in 10 mM MgCl$ sb2$, but is inhibited by both sulfate and pyrophosphate anions. The estimated K$ sb{ rm M}$ values were 2.3 $ mu$M for the oligonucleotide substrate and 11.8 $ mu$M for ATP. Similar to an enzymatic activity previously isolated from rat liver, SNQI is the first bovine preparation to display both 5$ sp prime$ kinase and 3$ sp prime$ phosphatase activities. / Partial purification and characterization of SNQII revealed similarities to SNQI, such as an acidic pH optimum and the presence of 3$ sp prime$ phosphatase activity. DNA kinase activity was also demonstrated in two mammalian cell lines.

Septins are a family of GTP-binding proteins implicated in mammalian cell division. Since these proteins form heterologous complexes and filaments in interphase cells, it has been assumed that depletion of any or all septins in a given cell type will give rise to the same phenotype. I demonstrate that while all septins expressed in HeLa cells localize to the cleavage furrow and midbody during cytokinesis, and co-immunoprecipitate throughout cell division, they do not all have identical roles during this process. Specific depletion of SEPT2 or SEPT11 caused defects in the early stages of cytokinesis, ultimately resulting in binucleation. Similar results were observed upon simultaneous depletion of all septins. In sharp contrast, SEPT9 was dispensable for the early stages of cell division, but was critical for the final separation of daughter cells. I demonstrate that SEPT9 mediates the localization of the vesicle-tethering exocyst complex to the midbody. Immunofluorescence microscopy suggests that SEPT9 may act to compartmentalize the exocyst at the site of abscission, analogous to the role performed by septins in Saccharomyces cerevisiae. I provide evidence that the N-terminal region of SEPT9, which is absent from the shorter SEPT9 isoforms, plays an important role in abscission. I describe a long-anticipated link between a mammalian septin and the cell cycle machinery by showing that the N-terminal region of SEPT9 is phosphorylated at threonine 24 upon mitotic entry by cyclin-dependent kinase 1. This creates a binding site for the WW domain of the peptidyl-prolyl isomerase Pin1. I provide evidence that Pin1 induces a conformational change in the N-terminal region of SEPT9 that is important for the completion of cytokinesis. I propose that mitotic regulation of SEPT9 by Cdk1 and Pin1 regulates an interaction between SEPT9 and an unidentified protein that is critical for abscission.

Septins are a family of GTP-binding proteins implicated in mammalian cell division. Since these proteins form heterologous complexes and filaments in interphase cells, it has been assumed that depletion of any or all septins in a given cell type will give rise to the same phenotype. I demonstrate that while all septins expressed in HeLa cells localize to the cleavage furrow and midbody during cytokinesis, and co-immunoprecipitate throughout cell division, they do not all have identical roles during this process. Specific depletion of SEPT2 or SEPT11 caused defects in the early stages of cytokinesis, ultimately resulting in binucleation. Similar results were observed upon simultaneous depletion of all septins. In sharp contrast, SEPT9 was dispensable for the early stages of cell division, but was critical for the final separation of daughter cells. I demonstrate that SEPT9 mediates the localization of the vesicle-tethering exocyst complex to the midbody. Immunofluorescence microscopy suggests that SEPT9 may act to compartmentalize the exocyst at the site of abscission, analogous to the role performed by septins in Saccharomyces cerevisiae. I provide evidence that the N-terminal region of SEPT9, which is absent from the shorter SEPT9 isoforms, plays an important role in abscission. I describe a long-anticipated link between a mammalian septin and the cell cycle machinery by showing that the N-terminal region of SEPT9 is phosphorylated at threonine 24 upon mitotic entry by cyclin-dependent kinase 1. This creates a binding site for the WW domain of the peptidyl-prolyl isomerase Pin1. I provide evidence that Pin1 induces a conformational change in the N-terminal region of SEPT9 that is important for the completion of cytokinesis. I propose that mitotic regulation of SEPT9 by Cdk1 and Pin1 regulates an interaction between SEPT9 and an unidentified protein that is critical for abscission.

The LD(_50) of 10-day-old blowflies differed significantly in two different stocks, and were found to be 38.12 ± 0.07ºC for the Durham stock and 40.8 ± 0.18ºC for the Cambridge stock. A transitory increase in heat resistance occurred following the exposure of adult blowflies to a sublethal heat shock. This thermotolerance was apparent 1h after the application of heat shock, was maximal 2-3 h later and had disappeared after 6 h. Oxidative phosphorylation by flight muscle mitochondria from non-thermotolerant control flies was impaired by an LD(_50) dose in vivo. Respiration using glycerol-3- phosphate was more heat sensitive than that with pyruvate plus proline. State III respiration was markedly inhibited, acceptor control (RCI) was lost with (G 3P) as substrate and so ADP:0 ratios were not measurable; whereas with pyruvate + proline as substrates, although State III respiration was inhibited by 50% and acceptor control was significantly reduced, ADP:0 remained measurable. Uncoupling of oxidative phosphorylation was obvious only with pyruvate + proline where State IV was significantiy increased. The development of thermotolerance protected oxidative phosphorylation against heat damage. With G-3-P respiration State III was largely restored and acceptor control was not significantly different from controls, but ADP:0 remained lower. With pyruvate + proline as substrates State III respiration was inhibited, but State IV was also lower without evidence of uncoupling of oxidative phosphorylation. Acceptor control was restored to control levels but ADP:0 values were lower. The lower ADP:0 ratios indicate some impairment of mitochondrial function occurred. The effect of experimental temperature in vitro on respiratory performance of mitochondria from non-pretreated control and thermotolerant LD(_50) flies was also determined between 19 and 39ºC. State III respiration was markedly temperature- dependent in mitochondria from non-pretreated control flies with both substrates; it was maximal at 24-29ºC and fell progressively at higher measuring temperatures. In mitochondria from thermotolerant flies, State III respiration was less temperature dependent with both substrates, but this effect was more marked for G-3-P. The effect of experimental temperature on State IV respiration was similar in mitochondria from non- pretreated control and thermotolerant LD(_50) flies with the same substrate, but differed between the two substrates. With G 3P as substrate, respiration rate rose with temperature with a Q(_10) of approximately 1.5; however, with pyruvate + proline as substrate, the trend was for respiration rate to fall as experimental temperature rose. Differences in the temperature sensitivities of mitochondria from control and thermotolerant flies, in terms of acceptor control, were found. Using G-3-P, acceptor control was lost in mitochondria from control flies above 29ºC, but was still measurable at 34ºC in mitochondria from thermotolerant flies. With pyruvate + proline as substrate acceptor control was demonstrable in mitochondria from both non-pre-treated control and thermotolerant flies at all experimental temperatures. The thermal sensitivities of the respiratory complexes were studied using the inhibitors rotenone and antimycin A. In mitochondria from LD(_50) treated control flies respiration uncoupled with FCCP was not restored to State II levels. However, in LD(_50) treated mitochondria from thermotolerant flies respiration uncoupled with FCCP was not different from State III respiration. These data suggest that the reduction in State III respiration after heating is owing to an inhibition of oxidation rather than phosphorylation. Complex I, NADH coenzyme Q reductase, was shown to be the most temperature sensitive of the respiratory complexes.

Thesis (M.S.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references (ℓ. 65-73)