PubMed · 5551561
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P T Englund, H O Smith, E Sandbek. 1971. A density gradient fractionator.. https://doi.org/10.1016/0003-2697(71)90412-x
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Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Ht) protein. A hallmark of HD is the proteolytic production of an N-terminal fragment of Ht, containing the polyQ repeat, that forms aggregates in the nucleus and cytoplasm of affected neurons. Proteins with longer polyQ repeats aggregate more rapidly and cause disease at an earlier age, but the mechanism of aggregation and its relationship to disease remain unclear. To provide a new, genetically tractable model system for the study of Ht, we engineered yeast cells to express an N-terminal fragment of Ht with different polyQ repeat lengths of 25, 47, 72, or 103 residues, fused to green fluorescent protein. The extent of aggregation varied with the length of the polyQ repeat: at the two extremes, most HtQ103 protein coalesced into a single large cytoplasmic aggregate, whereas HtQ25 exhibited no sign of aggregation. Mutations that inhibit the ubiquitin/proteasome pathway at three different steps had no effect on the aggregation of Ht fragments in yeast, suggesting that the ubiquitination of Ht previously noted in mammalian cells may not inherently be required for polyQ length-dependent aggregation. Changing the expression levels of a wide variety of chaperone proteins in yeast neither increased nor decreased Ht aggregation. However, Sis1, Hsp70, and Hsp104 overexpression modulated aggregation of HtQ72 and HtQ103 fragments. More dramatically, the deletion of Hsp104 virtually eliminated it. These observations establish yeast as a system for studying the causes and consequences of polyQ-dependent Ht aggregation.
We have cloned and inactivated in vivo, by repeat-induced point mutations, the nuclear gene encoding a 21 kDa subunit of complex I from Neurospora crassa. Mitochondria from the nuo21 mutant lack this specific protein but retain other subunits of complex I in approximately normal amounts. In addition, this mutant is able to assemble an almost intact enzyme. The electron transfer activities from NADH to artificial acceptors of mitochondrial membranes from nuo21 differ from those of the wild-type strain, suggesting that the absence of the 21 kDa polypeptide results in conformational changes in complex I. Nevertheless, complex I of nuo21 is able to perform NADH:ubiquinone reductase activity, as judged by the observation that the respiration of mutant mitochondria is sensitive to inhibition by rotenone. We discuss these findings in relation to the involvement of complex I in mitochondrial diseases.
Assembly of multi-subunit membrane protein complexes is poorly understood. In this study, we present direct evidence that the D1 protein, a multiple membrane spanning protein, assembles co-translationally into the large membrane-bound complex, photosystem II. During pulse-chase studies in intact chloroplasts, incorporation of the D1 protein occurred without transient accumulation of free labeled protein in the thylakoid membrane, and photosystem II subcomplexes contained nascent D1 intermediates of 17, 22, and 25 kDa. These N-terminal D1 intermediates could be co-immunoprecipitated with antiserum directed against the D2 protein, suggesting co-translational assembly of the D1 protein into PS II complexes. Further evidence for a co-translational assembly of the D1 protein into photosystem II was obtained by analyzing ribosome nascent chain complexes liberated from the thylakoid membrane after a short pulse labeling. Radiolabeled D1 intermediates could be immunoprecipitated under nondenaturing conditions with antisera raised against the D1 and D2 protein as well as CP47. However, when the ribosome pellets were solubilized with SDS, the interaction of these intermediates with CP47 was completely lost, but strong interaction of a 25-kDa D1 intermediate with the D2 protein still remained. Taken together, our results indicate that during the repair of photosystem II, the assembly of the newly synthesized D1 protein into photosystem II occurs co-translationally involving direct interaction of the nascent D1 chains with the D2 protein.