Mice homozygous for the fat mutation develop obesity and hyperglycaemia that can be suppressed by treatment with exogenous insulin. The fat mutation maps to mouse chromosome 8, very close to the gene for carboxypeptidase E (Cpe), which encodes an enzyme (CPE) that processes prohormone intermediates such as proinsulin. We now demonstrate a defect in proinsulin processing associated with the virtual absence of CPE activity in extracts of fat/fat pancreatic islets and pituitaries. A single Ser202Pro mutation distinguishes the mutant Cpe allele, and abolishes enzymatic activity in vitro. Thus, the fat mutation represents the first demonstration of an obesity-diabetes syndrome elicited by a genetic defect in a prohormone processing pathway.

Energy metabolism in humans is tuned to distinct sex-specific functions that potentially reflect the unique requirements in females for gestation and lactation, whereas male metabolism may represent a default state. These differences are the consequence of the action of sex chromosomes and sex-specific hormones, including estrogens and progesterone in females and androgens in males. In humans, sex-specific specialization is associated with distinct body-fat distribution and energy substrate-utilization patterns; i.e., females store more lipids and have higher whole-body insulin sensitivity than males, while males tend to oxidize more lipids than females. These patterns are influenced by the menstrual phase in females, and by nutritional status and exercise intensity in both sexes. This minireview focuses on sex-specific mechanisms in lipid and glucose metabolism and their regulation by sex hormones, with a primary emphasis on studies in humans and the most relevant pre-clinical model of human physiology, non-human primates.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze compartment-specific membrane fusion. Whereas most SNAREs are bona fide type II membrane proteins, Ykt6 lacks a proteinaceous membrane anchor but contains a prenylation consensus motif (CAAX box) and exists in an inactive cytosolic and an active membrane-bound form. We demonstrate that both forms are farnesylated at the carboxyl-terminal cysteine of the CCAIM sequence. Farnesylation is the prerequisite for subsequent palmitoylation of the upstream cysteine, which permits stable membrane association of Ykt6. The double-lipid modification and membrane association is crucial for intra-Golgi transport in vitro and cell homeostasis͞survival in vivo. The membrane recruitment and palmitoylation is controlled by the N-terminal domain of Ykt6, which interacts with the SNARE motif, keeping it in an inactive closed conformation. Together, these results suggest that conformational changes control the lipid modification and function of Ykt6. Considering the essential and central role of Ykt6 in the secretory pathway, this spatial and functional cycle might provide a mechanism to regulate the rate of intracellular membrane flow.T he dynamic and specific trafficking of proteins and lipids along the secretory and endocytic pathways relies on precisely choreographed membrane fusion events, in turn relying on the faithful pairing of cognate soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs) between membranes (1, 2). In vitro membrane fusion assays employing purified SNAREs reconstituted into liposomes, as well as in vivo cell-cell fusion experiments using f lipped SNAREs [cognate-vesicle (v) and target-membrane (t) SNAREs expressed on the extracellular surface of two cell populations, respectively], provide compelling evidence that SNAREs are the driving force for fusion and, in addition, encode targeting specificity (3-7). Furthermore, as reported for intraGolgi transport, some SNAREs function also as inhibitory SNAREs to fine-tune specific fusion events (8). Thus, the intracellular distribution of cognate SNAREs outlines the fusion potential of distinct compartments and provides a road map for membrane trafficking (9, 10).The rate at which compartments containing cognate SNAREs fuse is at least in part determined by the conformational state of SNAREs. Most SNAREs contain regulatory domains in addition to their SNARE motifs, which form the four-helix bundle at the core of the SNARE complex (11). These regulatory domains control the assembly of the three-helix t-SNARE (one helix derived from a syntaxin heavy chain and two from t-SNARE light chains) and͞or the subsequent v-͞t-SNARE complex formation. Additional components can either bind or posttranslationally modify SNAREs, increasing or decreasing SNARE activity.A unique and therefore interesting SNARE is Ykt6, an essential protein that is highly conserved from yeast to man (12). Ykt6 is special in that it is lipid-anchored to membranes, lacking the usual hydroph...

Syntaxin-5 (Sed5) is the only syntaxin needed for transport into and across the yeast Golgi, raising the question of how a single syntaxin species could mediate vesicle transport in both the anterograde and the retrograde direction within the stack. Sed5 is known to combine with two light chains (Bos1 and Sec22) to form the t-SNARE needed to receive vesicles from the endoplasmic reticulum. However, the yeast Golgi contains several other potential light chains with which Sed5 could potentially combine to form other t-SNAREs. To explore the degree of specificity in the choice of light chains by a t-SNARE, we undertook a comprehensive examination of the capacity of all 21 Sed5-based t-SNAREs that theoretically could assemble in the yeast Golgi to fuse with each of the 7 potential v-SNAREs also present in this organelle. Only one additional of these 147 combinations was fusogenic. This functional proteomic strategy thereby revealed a previously uncharacterized t-SNARE in which Sed5 is the heavy chain and Gos1 and Ykt6 are the light chains, and whose unique cognate v-SNARE is Sft1. Immunoprecipitation experiments confirmed the existence of this complex in vivo. Fusion mediated by this second Golgi SNAREpin is topologically restricted, and existing genetic and morphologic evidence implies that it is used for transport across the Golgi stack. From this study, together with the previous functional proteomic analyses which have tested 275 distinct quaternary SNARE combinations, it follows that the fusion potential and transport pathways of the yeast cell can be read out from its genome sequence according to the SNARE hypothesis with a predictive accuracy of about 99.6%.organelle ͉ syntaxin vesicle

Genetic and biochemical evidence has established that a SNARE complex consisting of syntaxin 5 (Sed5)-mYkt6 (Ykt6)-GOS28 (Gos1)-GS15 (Sft1) is required for transport of proteins across the Golgi stack in animals (yeast). We have utilized quantitative immunogold labeling to establish the cis-trans distribution of the v-SNARE GS15 and the t-SNARE subunits GOS28 and syntaxin 5. Whereas the distribution of the t-SNARE is nearly even across the Golgi stack from the cis to the trans side, the v-SNARE GS15 is present in a gradient of increasing concentration toward the trans face of the stack. This contrasts with a second distinct SNARE complex, also required for intra-Golgi transport, consisting of syntaxin 5 (Sed5)-membrin (Bos1)-ERS24 (Sec22)-rBet1 (Bet1), whose v-(rBet1) and t-SNARE subunits (membrin and ERS24), progressively decrease in concentration toward the trans face. Transport within the stack therefore appears to utilize countercurrent gradients of two Golgi SNAREpins and may involve a mechanism akin to homotypic fusion. INTRODUCTIONIntracellular membrane fusion results from the assembly of a target membrane t-SNARE, composed of one heavy chain (syntaxin) and two distinct light chains, with a cognate vesicle membrane v-SNARE (Sollner et al., 1993;Weber et al., 1998;Hu et al., 2003). The resulting SNAREpin, held together by a bundle of four ␣-helices (Sutton et al., 1998), forcefully perturbs the apposing lipid bilayers, thereby triggering fusion (McNew et al., 2000b). Cognate SNARE pairing between bilayers is the immediate determinant of the specificity of membrane fusion McNew et al., 2000a;Parlati et al., 2000Parlati et al., , 2002Paumet et al., 2001). The v-SNARE is functionally distinguished from the other subunits of the SNAREpin because fusion can only occur when the v-SNARE resides in the opposite bilayer from the remaining subunits, which comprise the t-SNARE .In particular, fusion of vesicles derived from the ER with the Golgi requires the pairing of the v-SNARE Bet1 (in yeast; rBet1 in animals) with the t-SNARE comprised of the syntaxin heavy chain Sed5 (syntaxin 5) and light chains Bos1 (membrin) and Sec22 (ERS24, also named Sec22b; , abbreviated t-Sed5/Bos1, Sec22. The genes encoding these four "ER-Golgi" SNAREs are required for ER-toGolgi transport in yeast in vivo (Newman and Ferro-Novick, 1987;Newman et al., 1990;Shim et al., 1991;Hardwick and Pelham, 1992;Cao and Barlowe, 2000). Liposomes bearing the v-SNARE Bet1 only fuse with liposomes bearing the cognate t-SNARE Sed5/Bos1, Sec22 (Parlati et al., , 2002Paumet et al., 2001).The intracellular distribution of these SNAREs determined by immunoelectron microscopy fits well with their roles in ER-to-Golgi transport in animal cells (Paek et al., 1997;Hay et al., 1998;Orci et al., 2000;Martinez-Menarguez et al., 2001). The "short" Golgi-localized form of the t-SNARE heavy chain syntaxin 5 (Hui et al., 1997), which lacks the ER-retrieval signal encoded in the additional exon of the "long" form, is located in every cisternae across the Golgi stack i...