PubMed · 13891778
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P FATT. 1961. Intracellular microelectrodes.. https://pubmed.ncbi.nlm.nih.gov/13891778/
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Many proteins of the secretory pathway contain disulfide bonds that are essential for structure and function. In the endoplasmic reticulum (ER), Ero1 alpha and Ero1 beta oxidize protein disulfide isomerase (PDI), which in turn transfers oxidative equivalents to newly synthesized cargo proteins. However, oxidation must be limited, as some reduced PDI is necessary for disulfide isomerization and ER-associated degradation. Here we show that in semipermeable cells, PDI is more oxidized, disulfide bonds are formed faster, and high molecular mass covalent protein aggregates accumulate in the absence of cytosol. Addition of reduced glutathione (GSH) reduces PDI and restores normal disulfide formation rates. A higher GSH concentration is needed to balance oxidative folding in semipermeable cells overexpressing Ero1 alpha, indicating that cytosolic GSH and lumenal Ero1 alpha play antagonistic roles in controlling the ER redox. Moreover, the overexpression of Ero1 alpha significantly increases the GSH content in HeLa cells. Our data demonstrate tight connections between ER and cytosol to guarantee redox exchange across compartments: a reducing cytosol is important to ensure disulfide isomerization in secretory proteins.
Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known. Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing. Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants. These mutations not only reveal a more precise function for rpS14 in ribosome biogenesis but also uncover a role in ribosome assembly for the extended tails found in many ribosomal proteins. These studies are one of the first to relate the structure of eukaryotic ribosomes to their assembly pathway-the carboxy-terminal extension of rpS14 is located in the 40S subunit near the 3' end of 18S rRNA, consistent with a role for rpS14 in 3' end processing of 20S pre-rRNA.
The filamentous fungi have dynamic and variable hyphal structures within which cytoplasm can be moved, synthesized, and degraded, in response to changes in environmental conditions, resource availability, and resource distribution. Their study has gone through several phases. In the first phase, direct observation was emphasized without undue concern for interior structures or in the presence of cytoplasm. By the mid-1970s, single biochemical proxies (ergosterol, marker fatty acids, chitin derivatives, etc.) were being used increasingly. The use of these surrogate single measurements continues, in spite of their inability to provide information on the physical structure of the filamentous fungi. Molecular approaches also are being used, primarily through the use of bulk nucleic acid extraction and cloning. Because the sources of the nucleic acids used in such studies usually are not known, taxonomic and phylogenetic information derived by this approach cannot be linked to specific fungal structures. Recently, a greater emphasis has been placed on assessing physical aspects of indeterminate fungal growth, involving the assessment of cytoplasm-filled and evacuated (empty) hyphae. Both of these parameters are important for describing filamentous fungal growth and function. The use of phase contrast microscopy and varied general stains, as well as fluorogenic substrates with observation by epifluorescence microscopy, has made it possible to provide estimates of cytoplasm-filled hyphal lengths. Using this approach, it has been possible to evaluate the responses of the indeterminate fungal community to changes in environmental conditions, including soil management. It is now possible to obtain molecular information from individual bacteria and fungal structures (hyphae, spores, fruiting bodies) recovered from environments, making it possible to link individual fungal structures with their taxonomic and phylogenetic information. In addition, this information can be considered in the context of the indeterminate filamentous fungal lifestyle, involving the dynamics of resource allocation to hyphal structural development and synthesis of cytoplasm. Use of this approach should make it possible to gain a greater appreciation of the indeterminate filamentous fungal lifestyle, particularly in the context of microbial ecology.