Insig regulates HMG-CoA reductase by controlling enzyme phosphorylation in fission yeast

Abstract

Insig functions as a central regulator of cellular cholesterol homeostasis by controlling activity of HMG-CoA reductase (HMGR) in cholesterol synthesis. Insig both accelerates the degradation of HMGR and suppresses HMGR transcription through the SREBP-Scap pathway. The fission yeast Schizosaccharomyces pombe encodes homologs of Insig, HMGR, SREBP, and Scap, called ins1(+), hmg1(+), sre1(+), and scp1(+). Here, we characterize fission yeast Insig and demonstrate that Ins1 is dedicated to regulation of Hmg1, but not the Sre1-Scp1 pathway. Using a sterol-sensing domain mutant of Hmg1, we demonstrate that Ins1 binding to Hmg1 inhibits enzyme activity by promoting phosphorylation of the Hmg1 active site, which increases the K(M) for NADPH. Ins1-dependent phosphorylation of Hmg1 requires the MAP kinase Sty1/Spc1, and Hmg1 phosphorylation is physiologically regulated by nutrient stress. Thus, in fission yeast, Insig regulates sterol synthesis by a different mechanism than in mammalian cells, controlling HMGR phosphorylation in response to nutrient supply.

Figure 1. Fission yeast Ins1 binds Hmg1 and is required for cellular lipid homeostasis

(A) Alignment of human and fission yeast Insig. Black bars denote previously determined (Insig1) or predicted (Ins1) transmembrane spans (Feramisco et al., 2004). (B) Ins1-TAP was purified from indicated strains grown in YES medium and samples were blotted with anti-Hmg1 IgG, anti-Myc IgG or rabbit IgG (to detect the TAP tag). Bound fraction is 10 times overloaded relative to unbound fraction. Asterisk denotes a product of in vitro proteolysis of Ins1-TAP. (C) Sterols were extracted from the indicated strain carrying either empty vector or overexpressing hmg1⁺ (nmt-hmg1⁺) and analyzed by gas chromatography. Values were normalized to wild-type cells. Error bars denote one standard deviation. Relative gas chromatograph signals for squalene, lanosterol and ergosterol in wild-type cells were 1, 14, and 31, respectively. (D) Extracts were prepared from cells grown in YES + 200 μg/ml cycloheximide and blotted using anti-Hmg1 or anti-Sre1 IgG. The precursor form of Sre1 is shown.

Figure 2. Ins1 inhibits Hmg1 activity

Microsomes from wild-type and ins1Δ cells were assayed for HMG-CoA reductase activity. Each data point represents the mean of 8 – 12 measurements. V_max and K_m are shown ± standard error of measurement. (E) Assayed microsomes were blotted using anti-Hmg1 IgG.

Figure 3. Hmg1 requires two conserved, adjacent intramembrane phenylalanines for Ins1 binding and enzyme inhibition

(A) Ins1 was immunoprecipitated from the indicated yeast strains. Bound (lanes 1–4) and input (lanes 5–8) fractions were blotted using anti-Hmg1 IgG or HRP-conjugated anti-Ins1 IgG. Bound fraction is 10 times overloaded relative to input fraction. (B) Microsomes were assayed for HMG-CoA reductase activity from ins1⁺ or ins1Δ cells expressing either hmg1⁺ or hmg1 F358A F359A. Microsomes (1.5 μg) were blotted using anti-Hmg1 IgG (B, bottom panel). (C) Ratio of HMGR activity between ins1Δ and ins1⁺ is given for indicated strains. Error bars denote the standard error.

Figure 4. Phosphorylation of Hmg1 S1024 and T1028 requires binding to Ins1 and is required for Ins1-dependent inhibition of Hmg1 activity

(A) Hmg1 was immunoprecipitated from the indicated strains labeled with ³²P H₃PO₄ for 3 h. Samples were analyzed by SDS-PAGE followed by autoradiography (top panel) or immunoblotting with anti-Hmg1 IgG (bottom panel). (B) BLAST alignment of fission yeast Hmg1 and human HMGR. Phosphorylated residues are boxed. (C) Hmg1 was immunoprecipitated from the indicated strains labeled with ³²P H₃PO₄ as in (A). (D and E) Hmg1 was immunoprecipitated from the indicated strains and samples were blotted using anti-Hmg1 IgG or phospho-specific antibodies. (F) Microsomes from indicated strains were assayed for HMG-CoA reductase activity and also blotted for Hmg1 (bottom panel). (G) Ratio of HMGR activity between ins1Δ and ins1⁺ is given for strains in (F). Error bars denote the standard error.

Figure 5. Sty1 is required for Hmg1 phosphorylation in response to nutrient supply

(A) Wild-type cells were grown in YES medium overnight. Cells were collected by centrifugation and resuspended in EMM. After 60 min, yeast extract (5 g/L) was added to the culture. Samples were taken at the indicated times, and Hmg1 immunoprecipitates were blotted using anti-Hmg1 IgG or phospho-specific antibodies. (B) Hmg1 was immunoprecipitated from the indicated strains cultured in EMM and samples were blotted using anti-Hmg1 IgG or phospho-specific antibodies. (C) sty1–12myc cells were grown in YES medium overnight. Cells were collected by filtration and resuspended in EMM, or were exposed to 0.6 M KCl for 30 min in YES. Protein samples taken at the indicated times were blotted with anti-phospho-p38 MAPK or anti-myc antibodies. (D) sty1-12myc, sty1Δ, and sty1-12myc ins1Δ cells were grown in YES in the absence or presence of 0.6 M KCl for 45 min. Hmg1 immunoprecipitates (top 3 panels) or whole-cell extracts (bottom panels) were immunoblotted using indicated antibodies.