Cytochrome b6 from isolated cytochrome b6f complexes Evidence for two spectral forms with different midpoint potentials

AbstractCytochrome b6 from spinach chloroplasts (either within the purified cytochrome b6f complex, or in its isolated form) exhibits two spectral species, which correspond to two midpoint potentials. This can be demonstrated by low temperature difference spectroscopy at fixed redox potentials. The high potential form of cytochrome b6 has a split α-peak at 557.5 and 561.5 nm, the low potential form has a symmetrical α-peak at 560.5 nm. Similar results were obtained with cytochrome b6 in the isolated cytochrome b6f complex from the cyanobacterium Anabaena variabilis

Molecular Architecture of the Yeast Nuclear Pore Complex: Localization of Nsp1p Subcomplexes

The nuclear pore complex (NPC), a supramolecular assembly of ∼100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the three- dimensional (3D) architecture of Xenopus NPCs, and eight of the ∼12 cloned and characterized vertebrate nucleoporins have been localized within the NPC. Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level. However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs. A direct comparison of yeast and Xenopus NPCs revealed that the NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions. With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket

The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix

AbstractThe cleavable prepiece of the precursor to yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) was attached to the amino-terminus of mouse dihydrofolate reductase (a cytosolic protein) by gene fusion. The resulting fusion protein was imported into the matrix of isolated, energized yeast mitochondria and cleaved to a polypeptide whose size was similar to that of authentic dihydrofolate reductase

Nucleocytoplasmic transport: factors and mechanisms

AbstractIn the past two years, our knowledge concerning the mechanisms of nucleocytoplasmic transport through the nuclear pore complex (NPC) has considerably expanded. The application of in vitro systems that reconstitute nuclear protein import has allowed the identification of cytosolic factors that are required for the import process. Microinjection into Xenopus oocytes and yeast genetic systems have provided interesting candidates for RNA export mediators. Functional and structural analysis of nucleoporins has demonstrated the involvement of NPC components in the transport process. Finally, new concepts have emerged such as the integration of the mechanisms of the nuclear protein import and RNA export reactions and the assembly of the transport machinery at specialised domains of the NPC

A Mitochondrial Presequence Can Transport a Chloroplast-encoded Protein into Yeast Mitochondria

Ribulose-1,5-bisphosphate carboxylase/oxygenase of chloroplasts contains eight large and eight small subunits. The small subunit is encoded by nuclear DNA, synthesized in the cytoplasm, and imported into chloroplasts. The large subunit is encoded by chloroplast DNA and synthesized within chloroplasts. We show in this communication that the large subunit of Chlamydomonas chloroplasts could be efficiently imported into isolated yeast mitochondria if it was attached to the presequence of a protein transported into the yeast mitochondrial matrix. Thus, synthesis of the large subunit within chloroplasts does not reflect the inability of this subunit to cross membranes. The same mitochondrial presequence could also transport the nuclear-encoded small subunit into yeast mitochondria. However, when the two types of subunits were coimported into mitochondria, they did not assemble with each other inside the heterologous organelle

The intracellular location of two aminoacyl-tRNA synthetases depends on complex formation with Arc1p

In yeast, two aminoacyl-tRNA synthetases, MetRS and GluRS, are associated with Arc1p. We have studied the mechanism of this complex formation and found that the non-catalytic N-terminally appended domains of MetRS and GluRS are necessary and sufficient for binding to Arc1p. Similarly, it is the N-terminal domain of Arc1p that contains distinct but overlapping binding sites for MetRS and GluRS. Localization of Arc1p, MetRS and GluRS in living cells using green fluorescent protein showed that these three proteins are cytoplasmic and largely excluded from the nucleus. However, when their assembly into a complex is inhibited, significant amounts of MetRS, GluRS and Arc1p can enter the nucleus. We suggest that the organization of aminoacyl-tRNA synthetases into a multimeric complex not only affects catalysis, but is also a means of segregating the tRNA- aminoacylation machinery mainly to the cytoplasmic compartment

Comparative localization of protein A tagged and endogenous yeast nuclear pore complex proteins by immunoelectron microscopy

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