Mutational analysis of the karmellae-inducing signal in Hmglp, a yeast HMG-CoA reductase isozyme

Deborah A. Profantt, Christopher J. Roberts, Robin L. Wright

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

In response to elevated levels of HMG-CoA reductase, an integral endoplasmic reticulum (ER) membrane protein, cells assemble novel ER arrays. These membranes provide useful models for exploration of ER structure and function, as well as general features of membrane biogenesis and turnover. Yeast express two functional HMG-CoA reductase isozymes, Hmg1p and Hmg2p, each of which induces morphologically different ER arrays. Hmg1p induces stacks of paired nuclear-associated membranes called karmellae. In contrast, Hmg2p induces peripheral ER membrane arrays and short nuclear-associated membrane stacks. In spite of their ability to induce different cellular responses, both Hmg1p and Hmg2p have similar structures, including a polytopic membrane domain containing eight predicted transmembrane helices. By examining a series of recombinant HMG-CoA reductase proteins, our laboratory previously demonstrated that the last ER-lumenal loop (Loop G) of the Hmg1p membrane domain contains a signal needed for proper karmellae assembly. Our goal was to examine the primary sequence requirements within Loop G that were critical for proper function of this signal. To this end, we randomly mutagenized the Loop G sequence, expressed the mutagenized Hmg1p in yeast, and screened for inability to generate karmellae at wild-type levels. Out of approximately 4000 strains with Loop G mutations, we isolated 57 that were unable to induce wild-type levels of karmellae assembly. Twenty-nine of these mutants contained one or more point mutations in the Loop G sequence, including nine single point mutants, four of which had severe defects in karmellae assembly. Comparison of these mutations to single point mutations that did not affect karmellae assembly did not reveal obvious patterns of sequence requirements. For example, both conservative and non-conservative changes were present in both groups and changes that altered the total charge of the Loop G region were observed in both groups. Our hypothesis is that Loop G serves as a karmellae-inducing signal by mediating protein-protein or protein-lipid interactions and that amino acids revealed by this analysis may be important for maintaining the proper secondary structure needed for these interactions. Copyright (C) 2000 John Wiley and Sons, Ltd.

Original languageEnglish (US)
Pages (from-to)811-827
Number of pages17
JournalYeast
Volume16
Issue number9
DOIs
StatePublished - Jun 30 2000

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Hydroxymethylglutaryl CoA Reductases
Endoplasmic Reticulum
Yeast
Isoenzymes
Yeasts
Membranes
Proteins
Nuclear Envelope
Point Mutation
Mutation
Oxidoreductases
Membrane Proteins
Lipids
Amino acids
Amino Acids
Defects

Keywords

  • Endoplasmic reticulum
  • HMG-CoA reductase
  • Karmellae
  • Membrane biogenesis

Cite this

Mutational analysis of the karmellae-inducing signal in Hmglp, a yeast HMG-CoA reductase isozyme. / Profantt, Deborah A.; Roberts, Christopher J.; Wright, Robin L.

In: Yeast, Vol. 16, No. 9, 30.06.2000, p. 811-827.

Research output: Contribution to journalArticle

Profantt, Deborah A. ; Roberts, Christopher J. ; Wright, Robin L. / Mutational analysis of the karmellae-inducing signal in Hmglp, a yeast HMG-CoA reductase isozyme. In: Yeast. 2000 ; Vol. 16, No. 9. pp. 811-827.
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