Regulation of the yeast CYT1 gene encoding cytochrome c1 by HAP1 and HAP2/3/4.

Mitochondrial biogenesis requires the coordinate induction of hundreds of genes that reside in the nucleus. We describe here a study of the regulation of the nuclear-encoded cytochrome c1 of the b-c1 complex. Unlike cytochrome c, which is encoded by two genes, CYC1 and CYC7, c1 is encoded by a single gene, CYT1. The regulatory region of the CYT1 promoter contains binding sites for the HAP1 and HAP2/3/4 transactivators that regulate CYC1. The binding of HAP1 to the CYT1 element was studied in detail and found to differ in two important respects from binding to the CYC1 element. First, while CYC1 contains two sites that bind HAP1 cooperatively, CYT1 has a single high-affinity site. Second, while the CYT1 site and the stronger HAP1-binding site of CYC1 share a large block of homology, the HAP1 footprints at these sites are offset by several nucleotides. We discuss how these differences in HAP1 binding might relate to the difference in the biology of cytochrome c and cytochrome c1.     

Multiple domains mediate heme control of the yeast activator HAP1

The activity of the yeast activator HAP1in vivo requires heme. A heme responsive domain of HAP1 was identified previously. It is adjacent to the DNA-binding domain, and appears to block DNA binding in the absence of heme. Here we describe a novel genetic selection which yielded mutants of HAP1 that are independent of heme when assayed for activation. These mutants define a second region of HAP1, close to the activation domain, which also controls its response to heme.     

Lysophosphatidic receptor, LPA3, is positively and negatively regulated by progesterone and estrogen in the mouse uterus

Reciprocal interactions between blastocysts and receptive uteri are essential for successful implantation. This process is regulated by the timely interplay of two ovarian hormones, progesterone and estrogen. However, the molecular targets of these hormones are largely unknown. We showed recently that a small bioactive lysophospholipid, lysophosphatidic acid, plays a pivotal role in the establishment of implantation via its cellular receptor, LPA(3). Here we demonstrate that LPA(3) expression is positively and negatively regulated by steroid hormones in mouse uteri. The LPA(3) mRNA level in the uteri increased during early pseudopregnancy, peaking around 3.5 days post coitus (3.5 d.p.c.), then, decreased to the basal level on 4.5 d.p.c. LPA(3) expression remained at a low level in ovariectomized mice, and administration of progesterone to ovariectomized mice up-regulated LPA(3) mRNA expression. In addition, simultaneous administration of estrogen counteracted the effect of progesterone. These results show that progesterone and estrogen cooperatively regulate LPA(3) expression, thereby contributing to the receptivity of uteri during early pregnancy.     

WNK4 kinase negatively regulates the surface expression of muscarinic M3 receptor

With-No-Lysine [K] 4 (WNK4) kinase regulates the surface expression of various ion transporters. Not only ion transporters but G-protein coupled receptors (GPCR) can function properly when their expression level is appropriate at the plasma membrane. In this study, we examined the role of WNK4 kinase in the regulation of muscarinic receptor 3 (M₃R) using physiological and biochemical experiments. Measurement of the pilocarpine-responsive [Ca²⁺]_i change demonstrated that WNK4 kinase decreased the activity of M₃R through its reduced surface expression. Kinase domain of WNK4 bound with the third intracellular region of M₃R whereas its negative regulation was independent on the kinase activity. Comparable to wild-type WNK4, kinase-inactive WNK4^D318A mutant also reduced the surface expression of M₃R, whereas the kinase domain of WNK4_1-441 failed to reduce the surface expression of M₃R. In accordance with surface biotinylation experiments, non-permeable immunostaining of M₃R also showed that M₃R surface expression is independent on the kinase activity of WNK4. Interestingly, comparison of the half life of total and surface M₃R revealed that only the half life of total M₃R, but not surface M₃R was decreased by WNK4 kinase. Nevertheless, the rate of decrease in surface M₃R always exceeded that of total M₃R. Taken together, these results suggest that WNK4 kinase negatively regulates the anterograde trafficking of M₃R through kinase-independent mechanism.     

SIRT1 deacetylates and positively regulates the nuclear receptor LXR

The NAD(+)-dependent deacetylase Sir2 regulates life span in lower eukaryotes. The mammalian ortholog SIRT1 regulates physiological processes including apoptosis, fat metabolism, glucose homeostasis, and neurodegeneration. Here we show that SIRT1 is a positive regulator of liver X receptor (LXR) proteins, nuclear receptors that function as cholesterol sensors and regulate whole-body cholesterol and lipid homeostasis. LXR acetylation is evident at a single conserved lysine (K432 in LXRalpha and K433 in LXRbeta) adjacent to the ligand-regulated activation domain AF2. SIRT1 interacts with LXR and promotes deacetylation and subsequent ubiquitination. Mutations of K432 eliminate activation of LXRalpha by this sirtuin. Loss of SIRT1 in vivo reduces expression of a variety of LXR targets involved in lipid metabolism, including ABCA1, an ATP-binding cassette (ABC) transporter that mediates an early step of HDL biogenesis. Our findings suggest that deacetylation of LXRs by SIRT1 may be a mechanism that affects atherosclerosis and other aging-associated diseases.     

The CUP2 gene product regulates the expression of the CUP1 gene, coding for yeast metallothionein.

The yeast CUP1 gene codes for a copper-binding protein similar to metallothionein. Copper sensitive cup1s strains contain a single copy of the CUP1 locus. Resistant strains (CUP1r) carry 12 or more multiple tandem copies. We isolated 12 ethyl methane sulfonate-induced copper sensitive mutants in a wild-type CUP1r parental strain, X2180-1A. Most mutants reduce the copper resistance phenotype only slightly. However, the mutant cup2 lowers resistance by nearly two orders of magnitude. We cloned CUP2 by molecular complementation. The smallest subcloned fragment conferring function was approximately 2.1 kb. We show that CUP2, which is on chromosome VII, codes for or controls the synthesis or activity of a protein which binds the upstream control region of the CUP1 gene on chromosome VIII. Mutant cup2 cells produced extremely low levels of CUP1-specific mRNA, with or without added copper ions and lacked a factor which binds to the CUP1 promoter. Integrated at the cup2 site, the CUP2 plasmid restored the basal level and inducibility of CUP1 expression and led to reappearance of the CUP1-promoter binding factor. Taken collectively, our data establish CUP2 as a regulatory gene for expression of the CUP1 metallothionein gene product.