Characterization of the sec1-1 and sec1-11 mutations

Mirko Brummer, K. Kivinen, Jussi Jäntti (Corresponding Author), Jaana Toikkanen, Hans Söderlund, Sirkka Keränen

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Abstract

Sec1 proteins are implicated in positive and negative regulation of SNARE complex formation. To better understand the function of Sec1 proteins we have identified the nature of the temperature‐sensitive mutations in sec1‐1 and sec1‐11. The sec1‐1 mutation changes a conserved glycine443 to glutamic acid. The sec1‐11 mutation changes a highly conserved arginine432 to proline. Based on homology and the crystal structure of the mammalian nSec1p, the corresponding amino acids localize to the 3b domain of nSec1p. Compared to the wild‐type Sec1p the mutant proteins are less abundant even at the permissive temperature. Thus, the R432P and G443E mutations may cause structural alterations that affect folding and make the mutant proteins more susceptible to degradation. The remaining part is sufficient for growth and protein secretion at 24°C and thus is likely to be properly folded. At 37°C the mutant proteins become non‐functional. In pulse–chase‐type experiments the newly synthesized Sec1‐1 and Sec1‐11 proteins decayed similarly with the wild‐type protein. Thus, the non‐functionality of the mutant proteins cannot be explained by denaturation‐induced degradation only. It is possible that the newly synthesized mutant proteins fold slowly and are susceptible to degradation before they have managed to fold and associate with other proteins. The mutant proteins were unable to interact with the Sec1p‐interacting proteins Mso1p and Sso2p in the two‐hybrid assay, even at the permissive temperature. These results localize sec1‐1 and sec1‐11 mutations to a domain of Sec1p and suggest a mechanism by which sec1‐1 and sec1‐11 cells become temperature‐sensitive.
Original languageEnglish
Pages (from-to)1525-1536
Number of pages12
JournalYeast
Volume18
Issue number16
DOIs
Publication statusPublished - 2001
MoE publication typeA1 Journal article-refereed

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Brummer, M., Kivinen, K., Jäntti, J., Toikkanen, J., Söderlund, H., & Keränen, S. (2001). Characterization of the sec1-1 and sec1-11 mutations. Yeast, 18(16), 1525-1536. https://doi.org/10.1002/yea.796