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.

The mechanism by which a cell translates a spatially oriented, extracellular signal into a change in morphology and behavior is the key to understanding many biological processes. In order to investigate this general phenomenon, I have studied the chemotactic response of human polymorphonuclear leukocytes (PMN's) to f-methionyl-leucyl-phenylalanine (fMLP). Stimulation of PMN's with fMLP produces a plethora of intracellular events, including increases in cytosolic Ca+2. PMN's are also morphologically and behaviorally polarized by stimulation with chemoattractant; the membrane components and cytosolic organelles of polarized PMN's become asymmetrically distributed. Polarization and subsequent orientation of PMN's in the direction of fMLP are steps which precede and are necessary for chemotaxis. I have chosen to examine the role of Ca+2, a ubiquitous second messenger, in the polarization of PMN's to fMLP. To accomplish this goal, Ca+2 has been measured in resting and polarized PMN's, utilizing the intracellular fluorescence of the Ca+2-sensitive dye, fura-2. Initial experiments have revealed a Ca+2-insensitive form of fura-2 associated with PMN's which, if uncorrected, would lead to erroneous measurements of [Ca+2]. I have suggested putative sources for the Ca+2-insensitive fluorescence in PMN's and have presented two methods for accurate calculation of [Ca+2] in spite of the additional component of fluorescence. As measured from the cell-associated fluorescence of fura-2, [Ca+2] increases without a detectable lag upon addition of fMLP to PMN's in suspension. The rise in [Ca+2] is associated with an increase in the percentage of cells which polarize to fMLP. The increases in [Ca+2] and in polarization are both directly related to increases in the concentration of chemoattractant. Inhibition of the rise in [Ca+2], by exposure of the human donor to aspirin or addition of EGTA to isolated cells, results in a concommitant reduction in the percentage of cells which polarize to fMLP. These findings are consistent with the hypothesis that Ca+2 acts as a second messenger in the pathway of transduction of the extracellular signal which results in polarization. However, addition of ionomycin, the Ca+2-selective ionophore, to PMN's did not induce polarization either in the presence or in the absence of fMLP. This result suggests that increases Ca+2, which appear to be necessary for polarization, are locally distributed within the fMLP-stimulated PMN. Examination of the subcellular distribution of Ca+2 using the digital imaging microscope reveals that Ca+2 is not uniformly distributed in the polarized PMN. Cells polarized by stimulation with fMLP often exhibit regional differences in [Ca+2] from front to tail. The magnitude and direction of the intracellular gradient varies among cells and suggests that within individual cells, the heterogeneity of [Ca+2] varies temporally and spatially as the cell chemotaxes. The results of the experiments conducted in this dissertation suggest that Ca+2 plays an important role as second messenger in fMLP-stimulated PMN's. I suggest that the morphological polarity of the chemotactic PMN is dependent upon the establishment and maintenance of an intracellular Ca+2 gradient.

The SRP and SRP receptor have long been recognized as essential components of the protein translocation machinery in higher eukaryotes. The biochemical studies discussed in this thesis demonstrate that the signal recognition particle (SRP) mediated transport of proteins across the mammalian endoplasmic reticulum requires the participation of guanine ribonucleotides, in a capacity distinct from their role in polypeptide elongation. The requirement for guanine ribonucleotides during translocation was detected by experimentally separating the synthesis and transport phases of the translocation reaction. Here, the initial targeting of ribosomes to the membrane required SRP and an SRP receptor, but not GTP. However, the insertion of the nascent chain into the membrane required the presence of both SRP and SRP receptor, as well as, GTP. Further biochemical characterization of the initially targeted translocation intermediate demonstrated that SRP remains bound to targeted nascent signal sequences, unless GTP is present. The SRP-receptor catalyzed displacement of SRP from ribosomes was GTP-dependent both with intact membranes and with the purified SRP receptor preparations. GTP specific binding localized to the α subunit of the receptor by photoaffinity labeling and by probing nitrocellulose blots of the receptor with GTP. In addition, an analysis of the α subunit primary sequence revealed elements which are similar, yet not identical, to guanine ribonucleotide binding site consensus sequence elements. These results, taken together, indicate that the SRP receptor represents a novel class of GTP binding protein and is responsible for the guanine ribonucleotide mediated displacement of SRP from nascent signal sequences. A more detailed biochemical investigation of the GTP hydrolysis cycle of the SRP receptor demonstrated that the affinity between SRP and the SRP receptor is substantially greater in the presence of bound GTP and that the subsequent hydrolysis of bound GTP by SRα is necessary to recycle SRP to the cytoplasm. Purified SRP receptor was shown to hydrolyze GTP slowly. However, the GTP hydrolysis rate was substantially increased when both the SRP receptor and SRP were present in equimolar quantity. SRP does not hydrolyze GTP under these assay conditions. Moreover, free SRP was found not to compete effectively with SRP-ribosome complexes for the receptor, implying that the conformation of SRP is altered upon binding to a signal sequence. This result suggests that the affinity between SRP and the SRP receptor may be exquisitely regulated in order to prevent futile GTP hydrolysis cycles from occurring in the absence of secretory protein synthesis. Furthermore, the demonstration that the SRP receptor is a GTP binding protein provides fundamental insight into the mechanism of protein translocation. The displacement of SRP appears to be tightly coupled to the membrane insertion of nascent signal sequences. The membrane inserted intermediate in nascent chain translocation can be characterized by i) a resistance to extraction from the membrane with either EDTA or 0.5M KOAc; ii) an insensitivity to protease digestion, even after dissolution of the membrane with nonionic detergent. These results indicate that SRP displacement allows the nascent chain to interact with an additional membrane bound, protein component of the cellular translocation apparatus. Once in contact with this additional component, the nascent chain is shown to be capable to transverse the membrane bilayer in the absence of ribonucleotide hydrolysis or the continued elongation of the polypeptide. Thus, the results are incompatible with postulated mechanisms of protein translocation requiring that energy be derived from the continued elongation of the nascent polypeptide or from the direct interaction of a hydrophobic signal sequence with the lipid bilayer.

The human immunodeficiency virus type-1 matrix (HIV-1 MA) is best described as a multi-functional, structural protein. However, the multitude of functional activities ascribed to this viral component is not nearly as interesting as are its seemingly paradoxical and opposing roles during the viral life cycle. At the time of virus infection, HIV-1 MA remains associated with the reverse transcription complex, in which viral nucleic acids are synthesized, and facilitates its translocation to the host cell nucleus (Bukrinsky, Sharova et al. 1992; Bukrinsky, Sharova et al. 1993). This activity of MA has been proposed to form the basis for the infection of non-dividing cells (Bukrinsky, Haggerty et al. 1993). An interaction between the C-terminally phosphorylated form of MA and HIV-1 integrase, an integral component of the complex, was initially proposed to mediate this association (Gallay, Swingler et al. 1995; Gallay, Swingler et al. 1995). However, conditions which promote dissociation of integrase from the reverse transcription complex do not reduce MA association (Miller, Farnet et al. 1997). The possibility of a direct interaction between MA and the viral genome is discussed in Chapter III. The nucleophilic nature of HIV-1 MA is paradoxical with its reported activity in targeting the viral precursor proteins to the cytoplasmic membrane (Krausslich and Welker 1996), during the particle production phase of the viral life cycle. Furthermore, MA when expressed in the absence of other viral proteins exhibits a cytoplasmic localization (Fouchier, Meyer et al. 1997); a result which does not support a nuclear translocation role for this protein. The work presented here resolves this seemingly controversial issue. We demonstrate that MA exhibits a strong nuclear export activity. This newly discovered activity is designed to effectively counteract the protein's innate nucleophilic nature, thus maintaining a cytoplasmic localization. The nuclear export function of MA is sensitive to changes within the conformation of the protein as C- and N-terminal deletions, as well as point mutations in the protein, abolish the activity. Furthermore, the export activity is mediated by the Crm1 NES receptor (Fornerod, Ohno et al. 1997; Fukuda, Asano et al. 1997; Ossareh-Nazari, Bachelerie et al. 1997) despite the lack of a leucine-rich export signal within the matrix coding region. Therefore, the interaction between matrix protein and Crm1 is most likely to be mediated by another, perhaps cellular, protein. Any changes in matrix structure may lead to the disruption of this protein-protein interaction. We discuss a model implicating a phosphorylation event in the inactivation of this nuclear export signal. An even more fascinating issue regards the role of this nuclear export activity, during the viral life cycle, and is detailed in Chapter II. In short, mutations in MA which impair its nuclear export activity result in nuclear accumulation of the precursor Gag polyprotein (Pr55) and the nucleocapsid-associated viral genomic RNA. As a result, non-infectious virions deficient in genomic viral RNA are produced. Therefore, drugs designed to block this export activity can undermine the carefully orchestrated course of events during HIV replication and can shut down the growth of the virus.

NF-кB/Rel family proteins regulate genes that are critical for many cellular processes including apoptosis, inflammation, immune response, as well as development. NF-кB/Rel proteins function as homodimers or heterodimers, which recognize specific DNA sequences within target promoters. I examined the activity of different Drosophila Rel-related proteins in modulating Drosophila immunity genes by expressing the Rel proteins in stably transfected cell lines. I also compared how different combinations of these transcriptional regulators control the activity of various immunity genes. The results show that Rel proteins are directly involved in regulating the Drosophila antimicrobial response. Furthermore, expression of drosomycin and defensin is best induced by the Relish/Dif and the Relish/Dorsal heterodimers, respectively; whereas attacin activity can be efficiently up-regulated by the Relish homodimer and heterodimers. These results illustrate how the formation of Rel protein dimers differentially regulates target gene expression. Another area of my research is to investigate the function of p38 MAP kinase (mitogen-activated protein kinase) in Drosophila immune response. In vertebrates, one of the responses evoked by the pro-inflammatory cytokines and lipopolysaccharide (LPS) is the initiation of a kinase cascade that leads to the phosphorylation of p38 MAP kinase on Thr and Tyr within the motif Thr-Gly-Tyr, which is located within subdomain VIII. Two genes that are highly homologous to the mammalian p38 MAP kinases were molecularly cloned and characterized. Furthermore, genes that encode two novel Drosophila MAP kinase kinases, D-MKK3 and D-MKK4, were identified. D-MKK3 is an efficient activator of both Drosophila p38 MAP kinases, while D-MKK4 is an activator of D-JNK but not D-p38. These data establish that Drosophila indeed possesses a conserved p38 MAP kinase signaling pathway. We have examined the role of the D-p38 MAP kinases in the regulation of insect immunity. The results revealed that one of the functions of D-p38 is to attenuate antimicrobial peptide gene expression induced by LPS.

Maternally inherited poly(A)+ RNAs are important for directing early development in many animal species. This thesis investigates the regulation of maternal mRNA in the South African clawed frog, Xenopus laevis. The first portion of this thesis examines an unusual class of maternal RNA, interspersed poly(A)+ RNA, which is composed of co-linear repeat and single copy sequences. A cDNA clone, called pXR, contains the repeat portion of an interspersed RNA that hybridizes to several different oocyte transcripts of diverse size that persist until the neurula stage. DNA sequence analysis of the cDNA and hybrid selection of the oocyte transcripts followed by in vitro translation show that molecules of this repeat family are not translatable. This data, combined with the developmental profile of XR containing RNAs, indicate that members of this repeat family are not likely to be maternal messenger RNAs. The second part of this thesis investigates the expression of a class of maternal mRNAs that are regulated by cytoplasmic polyadenylation during progesterone induced oocyte maturation. One particular mRNA G10, is stored as a polyadenylated RNA in the cytoplasm of stage VI oocytes until maturation when the process of poly(A) elongation stimulates its translation. Injection of mutant and wild-type mRNAs, synthesized in vitro, revealed that two sequence elements, UUUUUUAUAAAG and AAUAAA, were both necessary and sufficient for polyadenylation and polysomal recruitment of G10. Maturation promoting factor and cyclin as well as progesterone can induce polyadenylation but in each case protein synthesis is required. Extracts from oocytes and unfertilized eggs were employed to identify factors that may be responsible for maturation-specific polyadenylation. An 82 kd protein that binds to the UUUUUUAUAAAG in egg, but not oocyte extracts, was identified by UV crosslinking. This data suggests that p82 is a good candidate for a developmentally regulated protein that controls the expression of maternal messenger RNAs in early Xenopus development.

Enterohemorrhagic E. coli (EHEC) has emerged as an important agent of diarrheal disease in the developed countries. Attachment to host cells, an essential step during intestinal colonization by EHEC, is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed attaching and effacing (A/E) lesion, directly beneath bound bacteria. The outer membrane protein, intimin, is required for the formation of this structure, as is Tir, a bacterial protein that is translocated into the host cell and thought to function as a receptor for intimin. In this thesis, we characterized A/E lesion formation by in vivo and in vitro-grown EHEC, aimed at testing whether bacterial adaptation to the mammalian host included up regulation of A/E lesion formation. Our results showed that actin signaling by EHEC was induced upon bacterial growth in vivo, and this induction was likely due to the up regulation of multiple activities by in vivo-grown EHEC. We also focused on the interaction between intimin and the host cell, an interaction that triggers actin condensation of A/E lesion formation. We evaluated the role of β1 integrins, one of the proposed receptors of intimin, in A/E lesion formation, and demonstrated that β1 integrins are not essential for intimin-mediated cell binding and actin condensation. To better understand intimin function, we mapped the functional domains of intimin, showed that the minimal cell binding domain of intimin correlates with the minimal Tir-binding domain. This minimal Tir-binding domain, when purified and coated on latex beads, was sufficient to trigger actin condensation on preinfected mammalian cells, suggesting that Tir-binding by intimin is critical in the final step of A/E lesion formation. To further demonstrate the significance of the interaction between intimin and Tir in A/E lesion formation, we developed a yeast two-hybrid system to identify intimin mutants diminished in Tir-binding, and then characterized those mutants for the ability to trigger actin condensation, the final step of A/E lesion formation. Finally, as a first step to study the downstream actin signaling pathway after Tir-binding, we mapped the domain of Tir involved in intimin-binding, and showed that the N-terminus and C-terminus of Tir are likely to be localized in the host cell cytoplasm, available to interact with downstream effectors in actin signaling.

In the first part of this thesis, the expression of the Drosophila homolog (DER) of the vertebrate epidermal growth factor (EGF) receptor was investigated during development of the optic lobe in relation to cell division patterns. Patterns of Der expression were determined by in situ hybridization while the patterns of cell division were determined by incorporation of bromodeoxyuridine. Der transcripts within the CNS are located almost exclusively within a small subset of cells in the optic lobe. These cells represent the precursors of the lamina, the outermost synaptic region of the optic lobe. Laminal cells are postmitotic, being derived from the terminal cell divisions of ganglion mother cells in adjacent proliferation centers. Induction of Der expression coincides with the birth of laminal cells and continues through the first day after puparium formation, a time when extensive interactions between photoreceptor cell axons and developing laminal cells occur. By forty-eight hours postpupariation, the lamina is well developed and Der transcripts are no longer present. Mutant Der alleles do not affect the structure of the lamina in the larval stage but minor optic lobe defects are seen in adults bearing the Elp1 allele of the Der gene. These results suggest a non-mitotic role for DER in the development of the lamina. In the second part of this thesis, a P-element mutagenesis was carried out to identify second site mutations that suppress the Elp phenotype. Elp represents a hypermorphic mutation in the Der gene. Analysis of Elp protein by protein blotting indicates that the increased activity is not due to an increased level of gene expression. Three dominant mutations that suppress the Elp phenotype were identified and designated as Su(Elp)2, Su(Elp)3-1, and Su(Elp)3-2. Su(Elp)2 is a recessive embryonic lethal mutation which also affects viability of heterozygotes. Embryos collected from Su(Elp)2 parents have both cuticle and CNS defects. The cuticle is frequently missing or aberrant. The CNS is often hypertrophied and aberrant. Su(Elp)2 does not enhance Der loss of function mutations or suppress torRL3. Su(Elp)3-1 is a post-embryonic recessive lethal mutation. The Su(Elp)3-1 mutation reduces the severity of the eye pattern defects in Elp homozygotes and increases the viability of Elp homozygotes. In addition, Su(Elp)3-1 rescues the viablility of Elp/flb trans-heterozygotes. Su(Elp)3-1 is complex and may consist of multiple P-element insertions that act as suppressors of Elp. Su(Elp)3-2 also appears to be a complex suppressor mutation.

The immune system is a complex network of cells and molecules that is a powerful and necessary defense mechanism to protect the host from pathogens. When this system is non-functional or dysregulated, the host is susceptible to takeover or attack against self, both with often lethal sequelae. Over the past century remarkable advances have been made in understanding how the immune system functions and how to manipulate this knowledge for human benefit. One strategy used to understand immune system function is to determine how the activity of immune system cells is modulated by the proteins these cells express on their surface. One rat T cell surface protein which was originally identified with antibodies almost two decades ago is the rat T cell alloantigen, RT6. During the intervening time enormous progress has been made in understanding the function of RT6+ T cells in normal and abnormal immune responses. In addition, during this time the characterization of RT6 genes, proteins, and homologues has occurred. One characterization of RT6 that is enigmatically missing is the function of this molecule. With this information it would be possible to determine how this molecule modulates T cell function. Therefore this project set out to begin to functionally characterize RT6 proteins. Part 1 of this project set out to determine if cell-surface RT6 proteins, like some other T cell surface proteins, could mediate T cell activation. Part 2 of this project was based on the recent observation that RT6 is homologous to NAD-catabolizing enzymes, and it was investigated whether RT6 proteins have ADP-ribosyltransferase activity. In Part 1 of this work it is demonstrated that cell-surface RT6 proteins are capable of delivering activation signals to T cells. Crosslinking cell-surface RT6 with antibodies potentiates the ability of PMA treated T cells to proliferate in response to the T cell growth factors IL-2 and/or IL-4. Crosslinking RT6 on these cells increases the surface expression of IL-2 receptors, suggesting that RT6-mediated signals selectively enhance growth factor receptor expression. This work also investigated the mechanism through which RT6 may deliver its signal. It is demonstrated that RT6 proteins are physically associated with five other proteins, including the src family tyrosine kinases p56lck and p60fyn. This work also suggests a novel mechanism to regulate T cell signaling by accessory molecules, since PKC activation causes qualitative and quantitative changes in the proteins physically associated with RT6. This work indicates that cell-surface RT6 is capable of delivering an accessory T cell activation signal. Therefore, RT6 proteins may be involved in vivo with the activation and proliferation of RT6+ T cells. Previous work in another laboratory has demonstrated that the RT6.2 protein possesses NAD glycohydrolase activity and indicated that RT6 proteins share overall sequence homology with ADP-ribosyltransferases. In Part 2 of this work, RT6 proteins are shown to possess NAD:arginine ADP-ribosyltransferase activity. ADP-ribosylation of proteins is a modification known to affect cell signaling and function. It is further demonstrated in this work that the substrate for RT6, extracellular NAD, inhibits T cell proliferation in a dose- and stimulus-dependent manner. Taken together, these studies suggest that through their enzymatic activities RT6 proteins modulate T cell activity. This work is the first to demonstrate that RT6 has two, possibly separate, functional characteristics. RT6 can therefore be described as a bifunctional T cell surface protein. RT6+ T cells play critical roles in regulating immune system responses in health and disease. Because of these functional studies on RT6 proteins, it can now be investigated how RT6 proteins may modulate T cell responses in different immunological situations. Thus, this work will provide the foundation to determine if and how RT6 proteins modulate immune system function in health and disease.

The viability of multicellular aerobic organisms requires the binding and transport of molecular oxygen from the atmosphere to sites of metabolism. In the earthworm, Lumbricus terrestris, erythrocruorins are freely dissolved multi-subunit protein complexes that serve the same functions as red blood cells The aims of this study were to 1) determine the overall arrangement of hemoglobin chains and non-hemoglobin chains in Lumbricus erythrocruorin, 2) determine the stereochemical determinants specifying erythrocruorin's hierarchical symmetry, and 3) investigate the molecular and chemical basis for the remarkable cooperative binding of ligands to earthworm hemoglobin. Erythrocruorin is a highly cooperative oxygen-carrying protein with Hill coefficients measured at some pH's as high as n = 7.9. Crystallographic analysis of the whole erythrocruorin molecule structure to 5.5 Å resolution reveals a hierarchical organization of 144 oxygen-binding polypeptides and 36 non-hemoglobin linker polypeptide chains. The hemoglobin chains are arranged in a novel dodecameric substructure at the periphery of the complex, whereas 36 linker chains comprise the inner core and projected triple-stranded, helical coiled-coil spokes towards the center of the complex. Interdigitation of these spokes appears crucial for stabilizing the complex. Crystallographic analysis of crystals from isolated hemoglobin chains provides greater detail (resolution = 2.6 Å) and complete atomic models for the hemoglobin polypeptides. Comparison of these models with other hemoglobins reveal unique features in the distal heme pocket, including large aromatic residues at the B10 position in three of the four hemoglobin chains. Aromatic residues at this position have been implicated in other hemoglobins to confer resistance to oxidation. Molecular interactions across each subunit include pH-dependent interactions that are consistent with the observed Bohr effect on oxygen binding. Specifically a π-cation interaction between an arginine of one subunit to a histidine of the opposing subunit is likely an important molecular switch in the allosteric transition from a low to high affInity ligand-binding state.