Dual targeting of proteins to mitochondria and chloroplasts : Characterization of dual targeting peptides and their interaction with organellar receptors

Most mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins with an N-terminal targeting peptide (TP), which directs them to the correct organelle. There is also a group of proteins that are dual targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide (dTP). The aim of this thesis was to characterize dTPs with respect to physicochemical features, sequence patterns, structural properties and interaction with the mitochondrial and chloroplastic receptors. We have used different statistical methods, including a multivariate data analysis (MVDA) to analyse all available dTPs and compare them to organelle-specific TPs of proteome-identified mitochondrial and chloroplastic proteins from Arabidopsis thaliana. The overall amino acid sequence patterns of dTPs were intermediate between mitochondrial targeting peptides (mTPs) and chloroplastic targeting peptides (cTPs) but the greatest differences in amino acid composition were found within the very N-terminal region of dTPs, where especially arginines are highly overrepresented in relation to cTPs. Interestingly, introducing arginines to the dTPs showed clustering towards the mTPs in silico and resulted in inhibition of chloroplast import in vitro, suggesting that positive charges in the N-terminal region of TPs may function as an 'avoidance signal' for chloroplast import. Studies with the dTP of threonyl-tRNA synthetase (ThrRS-dTP) revealed that 60 amino acids were required to confer dual targeting. The purified ThrRS-dTP(2-60) inhibited import of organelle-specific proteins, providing evidence that dual and organelle-specific proteins use the same organellar import pathways. CD spectra indicated that ThrRS-dTP(2-60) has the propensity to form a-helical structure in membrane mimetic environments. Further, NMR investigations of interaction profiles of ThrRS-dTP(2-60) with the mitochondrial Tom20 and the chloroplastic Toc34 receptor demonstrated that the mode of the recognition of a dual targeting peptide by mitochondrial and chloroplastic receptors is different. Our studies provide thorough characterization of dTPs and present for the first time dTP-organellar receptor interactions on the molecular level.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.</p

Towards Institutional Stabilization and Development? : A Study of Inter-Organizational Cooperation in the Tajik Cotton Industry

Close to two decades after the break-up of the Soviet Union, there is still a marked difference in developmental paths, including institutional as well as economic development and performance among the states emerging from the ruins of the vast empire. Turning attention to the least successful post-Soviet region, Central Asia, and Tajikistan in particular, this thesis provides a contribution to the discussion of how to institutionalize social power and build the foundations for political community in post-colonial societies. It is argued here that increased institutional stability may be achieved through inter-organizational cooperation among main actors within an institutional setting. Through the dispersion of intra- and inter-organizational effects of cooperation beyond the action situations where they are produced, several goals may be achieved. These are increased predictability, transparency and durability in governance, a more equitable distribution of wealth, and (in relation to the kinship-foundation of Tajik society) the embracing of kin-divisions in society. The study examines how and why organizations decide to get involved in cooperative collective activities within the Tajik cotton industry, an industry infamous for its unscrupulous financing schemes to which local investors tie farmers; schemes lacking business ethics and the interconnection of the social and economic with political relations. The study, through an embedded case study of one project (the Farmers’ Ownership Model), also examines the institutional implications of inter-organizational collective activities. The study’s empirical base is a combination of data derived from literature, reports, reviews of official documents, as well as from interviews and an expert survey conducted among organizational representatives with expert knowledge on the Tajik cotton industry.                The results suggest that it is possible to divide the forms of cooperation into three broad categories; business based cooperation, development and support-based cooperation and unilateral cooperative activity. The latter category contains interactions based on helping as well as on coercion. The motives for cooperating follow the same divisions, with profit and position-related motives dominating business-based cooperation. Development and support-based cooperation are primarily motivated by non-profit factors, such as community-(re)building and knowledge enhancement, as well as position-related motives. Within the group of unilateral interactions, the same divisions valid for business-based cooperation (unilateral cooperative activity based on coercion) and development and support-based cooperation (unilateral cooperative activity based on helping) are found. The actors approached for this study confirm that the institutional setting is “difficult” and that the social and political climate is not supportive of inter-organizational cooperative collective activities. Despite this, the actors agree that the time is right for cooperative efforts. The implications of inter-organizational cooperative collective activities within the frameworks of the examined Farmers’ Ownership Model project are many and important from a stabilizing perspective. Numerous strategic effects have been identified, among which is the creation of the first open farmer-owned joint stock company in the country, providing crop financing, high quality input, and technical assistance to the farmers in the northern Sugd region.  This is the most tangible effect, as is legislative change related to the marketing of cotton. The learning and knowledge-creation effects as a result of inter-organizational interactions within the project are also substantial. Client as well as non-client farmers have, through the technical assistance provided through the project company, managed to improve awareness in terms of their legal status. In addition, the technical assistance component of the project has served to improve productivity and the quality of the cotton grown. Despite indications of attitudinal changes within the action situations examined, there is no clear-cut evidence of trust effects at the institutional level. The results of empirical examination of the Tajik cotton industry to a large extent support the central thesis of the study