Thesis (MTech (Electrical Engineering))--Peninsula Technikon, 2002. / Word processed copy. Summary in English. Includes bibliographical references (leaves 147-155). Also available online.

Raman microscopy is a tool used by physicists to collect molecular information from a wide variety of samples. Biophysicists have increasingly made use of Raman microscopy in combination with optical tweezers to identify the molecular makeup of structures inside cells. There are high levels of background and noise in Raman spectra from cells, however, that obscure low intensity scattering peaks and prevent complete molecular characterization. We have designed and built a Mechanical Modulation Raman Microscope(MMRM) that is capable of background subtraction and noise reduction for Raman spectra from cells in vivo. There are two mechanisms of modulation: (1) three-axis stage modulation for objects fixed to the coverslip and (2) separate optical trap modulation for objects in solution. In both cases, objects of interest are modulated in and out of the Raman excitation volume while spectra are collected. Difference spectra are created by subtracting the spectrum without the object from the spectrum including the object. These difference spectra are averaged over the number of cycles of modulation. With the mechanical modulation technique, the background in Raman spectra is removed, and the signal-to-noise ratio is improved by two orders of magnitude. This technique was applied to fission yeast cells. Mechanical modulation Raman spectra of exponentially growing cells and starved cells were collected in three dimensions, and spatial differences were observed in the molecular composition for different metabolic states of individual yeast cells. / text

A common maxim of protein biochemistry states, “structure is function.” This is generally just as true for an individual polypeptide chains as for multi-protein complexes. The advent of yeast tagged-protein libraries has allowed systematic screening of a protein’s local interaction partners as well as a roughly mapping its cellular location. Recently our group and others discovered hundreds proteins forming new structures in stationary phase yeast cells using the yeast GFP-tag library. That equates to well over a quarter of normally diffuse cytoplasmic proteins assembled into discrete structures that appear as foci or fibers, all of unknown function. This study provides evidence that many of these foci are formed by protein aggregation- that contrary the maxim, structure can be dysfunction. Furthermore, this study uses yeast to demonstrate the generality of cytoplasmic protein aggregation in response to a variety of stresses, provides evidence that increasing aggregation of particular cytoplasmic proteins correlates with aging even across organisms, and proposes a theoretical framework for how cellular energy levels affect protein aggregation propensity. / text

In budding yeast, the Trf4/5p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex recognizes unwanted RNA transcripts in the nucleus and then targets them to the nuclear exosome for rapid degradation, constituting an important pathway of nuclear RNA quality control. Each pre-mRNA splicing event unavoidably generates a RNA side-product that should be recognized by TRAMP and then removed by the nuclear exosome to prevent the potentially harmful sequestration of splicing factors and/or ribonucleotides. While successful pre-mRNA splicing inevitably produces a spliced-out intron, errors in pre-mRNA splicing lead to the emergence of either an abnormal splicing intermediate, or a splicing-incompetent pre-mRNA that cannot be properly spliced. However, it remains unclear how and when these RNA side-products of pre-mRNA splicing are recognized by TRAMP. In this study, chromatin immunoprecipitation (ChIP) was applied to demonstrate that both TRAMP and the nuclear exosome component Rrp6p are cotranscriptionally recruited to nascent RNA transcripts, particularly to intronic sequences, indicating that splicing side-products are recognized by TRAMP and committed to subsequent nuclear-exosome-mediated degradation in a cotranscriptional manner. Deletion of TRF4, of both AIR1 and AIR2, or of RRP6, resulted in accumulation of unspliced pre-mRNAs. Surprisingly, while such pre-mRNAs accumulated in rrp6 cells owing to defects in pre-mRNA degradation, the same phenotype in trf4 and air1air2 cells involved splicing defects, demonstrating that only TRAMP, but not the nuclear exosome, contributes to optimal pre-mRNA splicing. Consistent with a direct stimulatory role for TRAMP in pre-mRNA splicing, negative genetic interactions and physical interactions between Trf4p and several splicing factors were observed, and that Trf4p was further shown to be required for optimal recruitment of the splicing factor Msl5p. The direct facilitation of pre-mRNA splicing by TRAMP may act as a fail-safe mechanism to ensure the cotranscriptional recruitment of TRAMP to nascent intron-containing transcripts before or during pre-mRNA splicing, such that the subsequently generated spliced-out introns, abnormal splicing intermediates, or splicing-incompetent pre-mRNAs can be recognized immediately by TRAMP, and then targeted to the nuclear exosome for prompt degradation before their potentially harmful accumulation. Since most TRAMP and nuclear exosome components found in budding yeast also contain functional human homologs, this work provides important insights into how splicing side-products are rapidly degraded by the nuclear RNA quality control system in human cells, which have a much higher frequency of introns within their genome, and certainly require a much more efficient pathway for the removal of an increased amount of splicing side-products due to the greater number of splicing events. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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The Saccharomyces cerevisiae oxysterol binding protein homologue Kes1/Osh4 is a member of an enigmatic class of proteins found throughout Eukarya. This family of proteins is united by a ?-barrel structure that binds sterols and oxysterols. An N-terminal lid is thought to both sequester sterols inside the core and promote localization of Kes1 to regions of high membrane curvature via a predicted ArfGAP lipid packing sensor motif. Additionally, a phosphoinositide-binding region on a discrete surface of Kes1 has also been identified. In this thesis, structure-function analysis of Kes1 determined that phosphoinositide binding is required for membrane association in vitro, and in vivo phosphoinositide binding is required for localization to the Golgi. Ergosterol, the major sterol in S. cerevisiae, and membrane curvature had minimal effects on membrane association. This study also revealed a role for Kes1 in the regulation of both phosphatidylinositol-4-phosphate and phosphatidylinositol-3-phosphate homeostasis. Phosphoinositide and sterol binding by Kes1 are necessary for it to alter phosphatidylinositol-4-phosphate, but not phosphatidylinositol-3-phosphate homeostasis. Misregulation of phosphatidylinositol-4-phosphate homeostasis by Kes1 manifested itself in an inability of the v-SNARE Snc1 to traffic properly and was consistent with a defect in trans-Golgi/endosome trafficking. I went on to demonstrate a role for Kes1 in regulating the conversion of phosphatidylinositol-4-phosphate to phosphatidylinositol for the synthesis of sphingolipids, and I present a model for the role of Kes1 at the Golgi. Kes1 acts as a sterol sensor that regulates phosphatidylinositol-4-phosphate to sphingolipids metabolism, which ultimately regulates the delivery of proteins that assemble into lipid rafts for their transport from the Golgi to the plasma membrane. I also uncovered a previously unknown role for Kes1 in the regulation of the cytoplasm-to-vacuole and autophagy trafficking pathways, which is mediated by the ability of Kes1 to regulate phosphatidylinositol-3-phosphate homeostasis.

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