<p>One promising approach to balance the need for fertilizer and the welfare of the environment, is the development of plants that can fix atmospheric nitrogen independently. This innovation would not only reduce the need for synthetic fertilizers and manure but also help mitigate climate change and the nitrogen crisis. <strong>To this end, we first need to better study the nitroplast, how it interacts with the host organism and how it could be potentially introduced into other cells</strong>.</p>
<p>It has been discovered that, to ensure the endosymbiotic relationship, several proteins that are essential to UCYN-A are expressed in the host, <em>B. bigelowii</em>, and imported into the symbiont, similar to chloroplasts and mitochondria, though to a lesser extent <ahref="#cite11"style="color: #185A4F;">[11]</a>. Many of these proteins possess specialized localization peptides that direct their cellular function. In UCYN-A, these peptides are usually a C-terminal extension and are known as the “uTP” (UCYN-A Transit Peptide), although not yet identified <ahref="#cite11"style="color: #185A4F;">[11]</a>. Our first aim was to employ bioinformatics analyses to identify the characteristic <strong>motifs required for a protein to be imported by UCYN-A</strong>. For this, we made use of host (<em>B. bigelowii</em>) and nitroplast (UCYN-A) genome data as well as the proteomics data published by Coale <em>et al.</em>. <strong>We identified 2 putative uTP sequences with high likelihood, which we named uTP1 and uTP2</strong>.</p>
<p>To understand the functioning of the UCYN-A import mechanism, we attempted to <strong>identify the proteins involved in translocating</strong> host-encoded proteins into UCYN-A. First, we located genes in the host genome that are potentially involved in the translocation, based on their similarity to proteins in other import mechanisms such as from <em>Paulinella chromatophora</em> (UCYN-A analogue for photosynthesis). Potential chaperones analogous to heat-shock proteins were also included in the search. These chaperones are hypothesized to bind to proteins tagged by the uTP and keep them from folding, allowing translocation through the UCYN-A membrane. We then followed this by <strong>obtaining the tertiary structure of all candidate proteins</strong> using a structure prediction tool, and used <strong>docking</strong> tools to select candidate proteins likely to bind the previously identified transit motifs.</p>
<p>In addition to <em>in silico</em> experiments, we also aimed to investigate the transport mechanisms of UCYN-A <em>in vivo</em>. Instead of making use of plants as target organisms, we opted for using single-cell model eukaryote organisms, namely the yeast <em>S. cerevisiae</em> and the green alga <em>C. reinhardtii</em>. The initial <em>in vivo</em> characterization of the UCYN-A transport system involved <strong>examining the expression and localization of the UCYN-A transit peptides in these eukaryotic model organisms</strong> to test whether uTP would have any unexpected effect on cell viability and would not target any other organelle. To this end, we designed vectors, cloned them using Gibson-assembly, transformed bacteria, purified expression plasmids, and transformed those into <em>S. cerevisiae</em> and <em>C. reinhardtii</em>. We expressed uTP-tagged fluorescent proteins, together with controls targeting other organelles, and localization was assessed using fluorescence microscopy. Our preliminary results indicate that uTP did not target any other organelle and did not lead to alterations in cell morphology.</p>
