SummaryThermogenesis in brown adipose tissue (BAT) is fundamental to energy balance and is also relevant for humans. Bone morphogenetic proteins (BMPs) regulate adipogenesis, and, here, we describe a role for BMP8B in the direct regulation of thermogenesis. BMP8B is induced by nutritional and thermogenic factors in mature BAT, increasing the response to noradrenaline through enhanced p38MAPK/CREB signaling and increased lipase activity. Bmp8b−/− mice exhibit impaired thermogenesis and reduced metabolic rate, causing weight gain despite hypophagia. BMP8B is also expressed in the hypothalamus, and Bmp8b−/− mice display altered neuropeptide levels and reduced phosphorylation of AMP-activated protein kinase (AMPK), indicating an anorexigenic state. Central BMP8B treatment increased sympathetic activation of BAT, dependent on the status of AMPK in key hypothalamic nuclei. Our results indicate that BMP8B is a thermogenic protein that regulates energy balance in partnership with hypothalamic AMPK. BMP8B may offer a mechanism to specifically increase energy dissipation by BAT.

We performed a systematic mapping of interaction domains on COP I subunits to gain novel insights into the architecture of coatomer. Using the two-hybrid system, we characterize the domain structure of the a-, b ¢-, e-COP and b-, g-, d-, z-COP coatomer subcomplexes and identify links between them that contribute to coatomer integrity. Our results demonstrate that the domain organization of the b-, g-, d-, z-COP subcomplex and AP adaptor complexes is related. Through in vivo analysis of a-COP truncation mutants, we characterize distinct functional domains on a-COP. Its N-terminal WD40 domain is dispensable for yeast cell viability and overall coatomer function, but is required for KKXX-dependent traf®cking. The last~170 amino acids of a-COP are also nonessential for cell viability, but required for e-COP incorporation into coatomer and maintainance of normal e-COP levels. Further, we demonstrate novel direct interactions of coatomer subunits with regulatory proteins: b ¢-and g-COP interact with the ARF-GTP-activating protein (GAP) Glo3p, but not Gcs1p, and b-and e-COP interact with ARF-GTP. Glo3p also interacts with intact coatomer in vitro.

Coatomer is required for the retrieval of proteins from an early Golgi compartment back to the endoplasmic reticulum. The WD40 domain of ␣-COP is required for the recruitment of KKTN-tagged proteins into coatomer-coated vesicles. However, lack of the domain has only minor effects on growth in yeast. Here, we show that the WD40 domain of ␤ -COP is required for the recycling of the KTKLL-tagged Golgi protein Emp47p. The protein is degraded more rapidly in cells with a point mutation in the WD40 domain of ␤ -COP (sec27-95) or in cells lacking the domain altogether, whereas a point mutation in the Clathrin Heavy Chain Repeat (sec27-1) does not affect the turnover of Emp47p. Lack of the WD40 domain of ␤ -COP has only minor effects on growth of yeast cells; however, absence of both WD40 domains of ␣-and ␤ -COP is lethal. Two hybrid studies together with our analysis of the maturation of KKTN-tagged invertase and the turnover of Emp47p in ␣-and ␤ -COP mutants suggest that the two WD40 domains of ␣-and ␤ -COP bind distinct but overlapping sets of di-lysine signals and hence both contribute to recycling of proteins with di-lysine signals.

ADP‐ribosylation factors (ARFs) are critical regulators of vesicular trafficking pathways and act at multiple intracellular sites. ADP‐ribosylation factor‐GTPase‐activating proteins (ARFGAPs) are proposed to contribute to site‐specific regulation. In yeast, two distinct proteins, Glo3p and Gcs1p, together provide overlapping, essential ARFGAP function required for coat protein (COP)‐I‐dependent trafficking. In mammalian cells, only the Gcs1p orthologue, named ARFGAP1, has been characterized in detail. However, Glo3p is known to make the stronger contribution to COP I traffic in yeast. Here, based on a conserved signature motif close to the carboxy terminus, we identify ARFGAP2 and ARFGAP3 as the human orthologues of yeast Glo3p. By immunofluorescence (IF), ARFGAP2 and ARFGAP3 are closely colocalized with coatomer subunits in NRK cells in the Golgi complex and peripheral punctate structures. In contrast to ARFGAP1, both ARFGAP2 and ARFGAP3 are associated with COP‐I‐coated vesicles generated from Golgi membranes in the presence of GTP‐γ‐S in vitro. ARFGAP2 lacking its zinc finger domain directly binds to coatomer. Expression of this truncated mutant (ΔN‐ARFGAP2) inhibits COP‐I‐dependent Golgi‐to‐endoplasmic reticulum transport of cholera toxin (CTX‐K63) in vivo. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability. However, silencing all three ARFGAPs causes cell death. Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.

During meiotic maturation or after fertilization of invertebrate and vertebrate oocytes, many of the quiescent stored mRNAs are recruited into polysomes. In the clam, Spisula solidissima, such masked messages include the abundant mRNAs encoding cyclin A and the small subunit of ribonucleotide reductase. We have previously shown that mRNA-specific unmasking of these two messages can be achieved in vitro, in oocyte cell-free extracts, by the addition of antisense RNAs corresponding to a fairly short (130-140 nucleotides) segment in their cognate 3' untranslated regions. We postulated that the antisense RNAs prevented the binding of a masking repressor protein (Standart et al., 1990). Here we report UV-crosslinking and gel retardation studies which show that the masking portions of the translationally regulated mRNAs bind an oocyte protein of 82 kDa (p82), which is phosphorylated after fertilization. This modification was accompanied by altered RNP complex formation in gel retardation assays. These changes presumably reflect the activation of translation of the masked mRNAs. The role of p82 phosphorylation in maternal mRNA unmasking was assessed in a novel in vitro activation system developed from clam oocytes, based upon the natural rise in pH which accompanies fertilization. Concomitant with mRNA unmasking, several kinases, including cdc2 and MAP kinases were activated in this system, as was p82 phosphorylation. Inhibitors of serine/threonine kinases, including 6-DMAP, staurosporine, and H7 inhibited p82 phosphorylation, whereas inhibitors of tyrosine kinases, protein kinase C, cAMP-dependent protein kinase, and p70s6k did not prevent this modification. A specific inhibitor of cdc2 kinase, p27Kip1, prevented p82 phosphorylation and translational activation, strongly suggesting that p82 modification is required for unmasking.

SummaryAlthough many transcriptional pathways regulating BAT have been identified, the role of lipid biosynthetic enzymes in thermogenesis has been less investigated. Whereas cold exposure causes changes in the fatty acid composition of BAT, the functional consequences of this remains relatively unexplored. In this study, we demonstrate that the enzyme Elongation of Very Long Chain fatty acids 6 (Elovl6) is necessary for the thermogenic action of BAT. Elovl6 is responsible for converting C16 non-essential fatty acids into C18 species. Loss of Elovl6 does not modulate traditional BAT markers; instead, it causes reduced expression of mitochondrial electron transport chain components and lower BAT thermogenic capacity. The reduction in BAT activity appears to be counteracted by increased beiging of scWAT. When beige fat is disabled by thermoneutrality or aging, Elovl6 KO mice gain weight and have increased scWAT mass and impaired carbohydrate metabolism. Overall, our study suggests fatty acid chain length is important for BAT function.

Clam oocytes display a striking difference in the pattern of protein synthesis on fertilization, which is maintained when cell-free extracts are assayed in the rabbit reticulocyte lysate. The mRNAs encoding ribonucleotide reductase and cyclin A (the major masked messages in the oocyte extracts) can be translationally activated by gel filtration in 0.5 M KCl, presumably by removal of repressor protein(s). When synthetic RNAs corresponding to different segments of ribonucleotide reductase and cyclin A mRNAs were added to oocyte extracts in 0.5 M KCl in a "competitive unmasking" assay, specific and complete unmasking of these mRNAs occurred on addition of antisense 3'-noncoding transcripts. The unmasking region in ribonucleotide reductase mRNA maps to a region of 134 nucleotides centered one-third of the way down the 3'-noncoding region.

In the transcriptionally inert maturing oocyte and early embryo, control of gene expression is largely mediated by regulated changes in translational activity of maternal mRNAs. Some mRNAs are activated in response to poly(A) tail lengthening; in other cases activation results from de-repression of the inactive or masked mRNA. The 39 UTR cis-acting elements that direct these changes are defined, principally in Xenopus and mouse, and the study of their trans-acting binding factors is just beginning to shed light on the mechanism and regulation of cytoplasmic polyadenylation and translational masking. In the marine invertebrate, Spisula solidissima, the timing of activation of three abundant mRNAs (encoding cyclin A and B and the small subunit of ribonucleotide reductase, RR) in fertilized oocytes correlates with their cytoplasmic polyadenylation. However, in vitro, mRNA-specific unmasking occurs in the absence of polyadenylation. In Walker et al. (in this issue) we showed that p82, a protein defined as selectively binding the 39 UTR masking elements, is a homolog of Xenopus CPEB (cytoplasmic polyadenylation element binding protein). In functional studies reported here, the elements that support polyadenylation in clam egg lysates include multiple U-rich CPE-like motifs as well as the nuclear polyadenylation signal AAUAAA. This represents the first detailed analysis of invertebrate cis-acting cytoplasmic polyadenylation signals. Polyadenylation activity correlates with p82 binding in wild-type and CPE-mutant RR 39 UTR RNAs. Moreover, since anti-p82 antibodies specifically neutralize polyadenylation in egg lysates, we conclude that clam p82 is a functional homolog of Xenopus CPEB, and plays a positive role in polyadenylation. Anti-p82 antibodies also result in specific translational activation of masked mRNAs in oocyte lysates, lending support to our original model of clam p82 as a translational repressor. We propose therefore that clam p82/CPEB has dual functions in masking and cytoplasmic polyadenylation.