Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 111-124).

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 16, 2009). Includes bibliographical references.

Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance mechanism conserved in eukaryotes. This thesis explores several interesting aspects of the NMD pathway. One important aspect of NMD which is presently the subject of intense controversy is the subcellular localization of NMD. In one set of experiments, the decay kinetics of the ade2-1 and pgk1 nonsense mRNAs (substrates for NMD) were investigated in response to activating the NMD pathway to determine if cytoplasmic nonsense mRNAs are immune to NMD in the yeast system. The results of these studies demonstrated that activation of NMD caused rapid and immediate degradation of both the ade2-1 and the early nonsense pgk1 steady state mRNA populations. The half lives of the steady state mRNA populations for both ade2-1 and pgk1 (early nonsense) were shortened from >30 minutes to approximately 7 minutes. This was not observed for pgk1mRNAs that contained a late nonsense codon demonstrating that activation of NMD specifically targeted the proper substrates in these experiments. Therefore, in yeast, nonsense mRNAs residing in the cytoplasm are susceptible to NMD. While these findings are consistent with NMD occurring in the cytoplasm, they do not completely rule out the possibility of a nuclear-associated decay mechanism. To investigate the involvement of the nucleus in NMD, the putative nuclear targeting sequence identified in Nmd2p (one of the trans-acting factors essential for NMD) was characterized. Subcellular fractionation experiments demonstrated that the majority of Nmd2p localized to the cytoplasm with a small proportion detected in the nucleus. Specific mutations in the putative nuclear localization signal (NLS) of Nmd2p were found to have adverse effects on the protein's decay function. These effects on decay function, however, could not be attributed to a failure in nuclear localization. Therefore, the residues that comprise the putative NLS of Nmd2p are important for decay function but do not appear to be required for targeting the protein to the nucleus. These results are in accordance with the findings above which implicate the cytoplasm as an important cellular compartment for NMD. This thesis then investigates the regulatory roles of the trans-acting factors involved in NMD (Upf1p, Nmd2p, and Upf3p) using a novel quantitative assay for translational suppression, based on a nonsense allele of the CAN1 gene (can1-100). Deletion of UPF1, NMD2, or UPF3 stabilized the can1-100 transcript and promoted can1-100 nonsense suppression. Changes in mRNA levels were not the basis of suppression, however, since deletion of DCP1 or XRN1 or high-copy can1-100 expression in wild-type cells caused mRNA stabilization similar to that obtained in upf/nmd cells but did not result in comparable suppression. can1-100 suppression was highest in cells harboring a deletion of UPF1, and overexpression of UPF1 in cells with individual or multiple upf/nmd mutations lowered the level of nonsense suppression without affecting the abundance of the can1-100 mRNA. These findings indicate that Nmd2p and Upf3p regulate Upf1p activity and that Upf1p plays a critical role in promoting termination fidelity that is independent of its role in regulating mRNA decay.

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC) respectively act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the non-selective interaction between the ribosome and the Sec61 complex. The Sec61 complex, serving as both the high affinity ribosome receptor and the translocation channel, is performing two very different functions which presumably requires different activity domains within the Sec61 complex. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the N and C-terminus of Sec61α was used to map the location of proteolysis cleavage sites. We observed a striking correlation between a loss of ribosome binding activity and the digestion of the C-terminal tail or cytoplasmic loop 8 of Sec61α. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether ribosome binding to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind ribosomes with high affinity at physiological ionic strength remain active in SRP-independent translocation indicating that ribosome binding and translocation promotion are separable activities of the Sec61 complex. Translocation promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.

Translocation across or integration into the rough endoplasmic reticulum (RER) membrane is the first step in the intracelluar sorting of proteins in eukaryotic cells. This process is initiated when a signal sequence in the nascent protein chain emerges from the ribosome and is recognized by the signal recognition particle (SRP). The resulting SRP-ribosome-nascent chain-complex (SRP-RNC) is targeted to the RER membrane through the concerted action of the SRP and the SRP receptor (SR). The nascent chain is then displaced from SRP and transferred to the translocon, a proteinaceous channel composed of oligomers of the Sec61 complex. To gain a better understanding of the molecular mechanism of protein translocation, we treated ribosome-stripped micro somes with proteases of different cleavage specificities to sever cytoplasmic domains of SRα, SRβ, TRAM, and the Sec61 complex, and then characterized protein translocation intermediates that accumulate when Sec61α or SRβ is inactivated by proteolysis. We found that GTP hydrolysis by the SRα-SRP complex and dissociation of SRP54 from the signal sequence are blocked in the absence of a functional Sec61 complex. Experiments using SR-reconstituted proteoliposomes confirmed the assembly of a membrane-bound, GTP-stabilized post-targeting intermediate. These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54. This regulatory role of Sec61α is proposed to provide a mechanism that inhibits signal sequence dissociation from SRP54 if the adjacent Sec61 complex is occupied by a translating ribosome, thereby insuring efficient transfer of an RNC from the SRP-SR complex to the translocation channel. We also found that complex formation between SRα and SRP is compromised in the absence of intact SRβ. Results obtained using a soluble system of in vitro translated SRα and SRβ suggest that SRβ is either required for GTP binding to SRα and SRP54 or for stabilizing the SRα-SRP complex. Moreover, using the XTP mutants of SRα and SRP54, we found that XTP cannot support efficient protein translocation in the absence of GTP. The addition of GTP dramatically promotes protein translocation into the endoplasmic reticulum, suggesting the GTPase activity of SRβ is required for this process. Further mutagenesis experiments revealed that the GTP-binding pocket of SRβ is involved in dimerization with SRa. All these data demonstrate that SRβ is important in protein translocation and will help elucidate the precise role of SRβ in vivo.

<p>Type 1 diabetes mellitus affects millions of people worldwide. Islet transplantation is a minimal invasive surgical procedure that restores euglycemia and halts the progression of diabetic complications. However, despite transplantation of islets from multiple donors most patients reverse to hyperglycemia within five years. New strategies to improve long-term outcome of islet transplantation are indispensable. This thesis studied differences in the microvasculature between endogenous and transplanted pancreatic islets, and investigated means to improve islet graft revascularization and function. Islet graft microvessels were similar to endogenous islets responsive to adenosine, angiotensin II and nitric oxide (NO). Recipient hyperglycemia induced a higher basal islet graft blood flow, which also was less dependent on NO than in normoglycemic recipients. Transplantation of freshly isolated instead of cultured islets improved graft revascularization, oxygenation and function. Pretreatment of islets with vascular endothelial growth factor decreased their expression of matrix metalloproteinase-9 (MMP-9) and impaired graft revascularization. Moreover, MMP-9 pretreatment <i>per se</i> improved graft revascularization. <i>In vivo</i>, 20-25% of all endogenous rat islets was low oxygenated (pO₂ <10 mmHg). Changes in the islet mass, by means of whole-pancreas transplantation, doubled the fraction of low oxygenated islets in the endogenous pancreas of transplanted animals, whereas this fraction almost completely disappeared after a 60% partial pancreatectomy. Interestingly, oxygenation was related to metabolism, since well oxygenated islets <i>in vivo</i> had 50% higher leucine-dependent protein biosynthesis, which includes (pro)insulin biosynthesis. In intraportally transplanted islets, the low oxygenated fraction of islets was markedly increased one day post-transplantation, and the oxygenation remained low following revascularization. In summary, these data suggest that a better revascularization of transplanted islets can improve graft function. Furthermore, the oxygenation and metabolism of endogenous islets is tightly regulated. This regulation seems to be disturbed following transplantation, which may contribute to long-term islet graft failure. </p>

Type 1 diabetes mellitus affects millions of people worldwide. Islet transplantation is a minimal invasive surgical procedure that restores euglycemia and halts the progression of diabetic complications. However, despite transplantation of islets from multiple donors most patients reverse to hyperglycemia within five years. New strategies to improve long-term outcome of islet transplantation are indispensable. This thesis studied differences in the microvasculature between endogenous and transplanted pancreatic islets, and investigated means to improve islet graft revascularization and function. Islet graft microvessels were similar to endogenous islets responsive to adenosine, angiotensin II and nitric oxide (NO). Recipient hyperglycemia induced a higher basal islet graft blood flow, which also was less dependent on NO than in normoglycemic recipients. Transplantation of freshly isolated instead of cultured islets improved graft revascularization, oxygenation and function. Pretreatment of islets with vascular endothelial growth factor decreased their expression of matrix metalloproteinase-9 (MMP-9) and impaired graft revascularization. Moreover, MMP-9 pretreatment per se improved graft revascularization. In vivo, 20-25% of all endogenous rat islets was low oxygenated (pO2 &lt;10 mmHg). Changes in the islet mass, by means of whole-pancreas transplantation, doubled the fraction of low oxygenated islets in the endogenous pancreas of transplanted animals, whereas this fraction almost completely disappeared after a 60% partial pancreatectomy. Interestingly, oxygenation was related to metabolism, since well oxygenated islets in vivo had 50% higher leucine-dependent protein biosynthesis, which includes (pro)insulin biosynthesis. In intraportally transplanted islets, the low oxygenated fraction of islets was markedly increased one day post-transplantation, and the oxygenation remained low following revascularization. In summary, these data suggest that a better revascularization of transplanted islets can improve graft function. Furthermore, the oxygenation and metabolism of endogenous islets is tightly regulated. This regulation seems to be disturbed following transplantation, which may contribute to long-term islet graft failure.

Thesis (Ph.D. in Biophysics & Genetics, Human Medical Genetics Program) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 123-147). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Cotranslational translocation is initiated by targeting of a ribosome-bound nascent polypeptide chain (RNC) to the endoplasmic reticulum (ER) membrane. The targeting reaction is coordinated by the signal recognition particle (SRP) through its interaction with the RNC and the membrane-bound SRP receptor (SR). A vacant translocon is a prerequisite for the subsequent nascent chain release from SRP-SR-RNC complex. It has been proposed that the protease-accessible cytosolic domains of the Sec61p complex play an important role in posttargeting steps by providing the binding site for the ribosome or interacting with the SR to initiate the signal sequence releasing. In this study, we have investigated the detailed mechanism that allows transfer of the ribosome-nascent chain (RNC) from the SRP-SR complex to the translocon using yeast S. cerevisiaeas the model system. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in translocation of nascent polypeptides that utilize the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome binding activity, indicating that the L6 and L8 sec61mutants impact different steps in the cotranslational translocation pathway. An interaction between the signal recognition particle receptor (SR) and the Sec61 complex has been proposed to facilitate transfer of the ribosome-nascent chain (RNC) complex to an unoccupied translocon. The slow growth and cotranslational translocation defects caused by deletion of the transmembrane span of yeast SRβ (srp102pΔTMD) are exaggerated upon disruption of the SSH1 gene, which encodes the pore subunit of a cotranslational translocation channel. Disruption of the SBH2 gene, which encodes the β-subunit of the Ssh1p complex, likewise causes a synthetic growth defect when combined with srp102pΔTMD. The in vivo kinetics of translocon gating by RNCs were slow and inefficient in the ssh1Δ srp102pΔTMD mutant. A critical role for translocon β-subunits in SR recognition is supported by the observation that deletion of both translocon β-subunits causes a block in the cotranslational targeting pathway that resembles elimination of either subunit of the SR, and could be partially suppressed by expression of carboxy-terminal Sbh2p fragments.

The proper workings of an organism rely on the accurate expression of genes throughout its lifetime. An important determinant for protein production is the availability of template mRNA molecules, the net effect of which is governed by their rates of synthesis vs. their rates of degradation. Normal mRNAs are proposed to be relatively stable in the cytoplasm while present in a protective, circularized conformation – the closed loop – through eIF4G-bridged interactions with 3’-bound poly(A) binding protein (Pab1p) and 5’-bound eIF4E. Introduction of a premature nonsense codon into an otherwise normal mRNA results in its rapid destabilization in cells, suggesting that not all stop codons behave the same, and events at premature termination events that lead to accelerated degradation of nonsense-containing mRNAs likely differ from those at normal termination, in which normal decay rates are maintained. The enhanced degradation observed for nonsense-containing mRNAs occurs through an evolutionarily conserved pathway involving the products of the UPF1, UPF2/NMD2, and UPF3 genes, the precise biochemical roles of which have remained elusive. We have developed a yeast cell-free translation system that allows us to assay biochemical events occurring at premature termination codons, compare them to those occurring at normal terminators, and study the role of Upf1p in these events. We find that premature termination is an inefficient process compared to normal termination and that one outcome of termination at a premature termination codon (PTC) is reinitiation at a nearby start codon. This in vitro post-termination reinitiation phenotype is dependent on the presence of Upf1p, a finding we have recapitulated in vivo. We also developed biochemical assays to define a role for Upf1p in translation following premature termination in vitro and find that Upf1p is involved in post-termination ribosome dissociation and reutilization. Supporting this idea are our findings that Upf1p predominantly cosediments with purified 40S ribosomal subunits. Finally, using our in vitro translation/toeprinting system, we have further characterized events leading to the formation of the mRNA closed loop structure and find that two states of the closed loop exist. The first requires the preinitiation 48S complex and includes Pab1p, eIF4G, eIF4E, and eIF3, whereas the second is formed after 60S joining and additionally requires the translation termination factors eRF1 and eRF3.