N-Terminal modifications of the 19S regulatory particle subunits of the yeast proteasome |
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Authors: | Kimura Yayoi Saeki Yasushi Yokosawa Hideyoshi Polevoda Bogdan Sherman Fred Hirano Hisashi |
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Institution: | Kihara Institute for Biological Research/Graduate School of Integrated Science, Yokohama City University, Maioka 641-12, Totsuka, Yokohama, Japan |
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Abstract: | The yeast (Saccharomyces cerevisiae) contains three N-acetyltransferases, NatA, NatB, and NatC, each of which acetylates proteins with different N-terminal regions. The 19S regulatory particle of the yeast 26S proteasome consists of 17 subunits, 12 of which are N-terminally modified. By using nat1, nat3, and mak3 deletion mutants, we found that 8 subunits, Rpt4, Rpt5, Rpt6, Rpn2, Rpn3, Rpn5, Rpn6, and Rpn8, were NatA substrates, and that 2 subunits, Rpt3 and Rpn11, were NatB substrates. Mass spectrometric analysis revealed that the initiator Met of Rpt2 precursor polypeptide was processed and a part of the mature Rpt2 was N-myristoylated. The crude extracts from the normal strain and the nat1 deletion mutant were similar in chymotrypsin-like activity in the presence of ATP in vitro and in the accumulation level of the 26S proteasome. These characteristics were different from those of the 20S proteasome: the chymotrypsin-like activity and accumulation level of 20S proteasome were appreciably higher from the nat1 deletion mutant than from the normal strain. |
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Keywords: | Saccharomyces cerevisiae Proteasome 19S regulatory particle N-acetylation N-myristoylation Two-dimensional polyacrylamide gel electrophoresis Mass spectrometry |
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