Transport vesicles derived from the Golgi apparatus are thought to mediate biosynthetic transport across the Golgi stack. These vesicles are surrounded by a protein coat whose principal constituents are coatomer (a complex of seven distinct subunits or COPs) and ADP-ribosylation factor (ARF, an N-myristylated small GTP-binding protein). The coat proteins of the COP-coated vesicles were originally defined by ultrastructural criteria, however, and it is possible that important but minor coat proteins or cytoplasmic proteins needed for coat assembly may have been overlooked. Here we show that coatomer and ARF are the only cytoplasmic proteins needed for the assembly and budding of COP-coated vesicles. COP-coated buds may therefore form essentially by self-assembly from Golgi cisternae after an initial step in which GTP is used to allow ARF binding.

Modified tetrapyrroles such as chlorophyll, heme, siroheme, vitamin B 12 , coenzyme F 430 , and heme d 1 underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d 1 heme. The initial step in these transformations involves the decarboxylation of siroheme to give didecarboxysiroheme. For d 1 heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxysiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for siroheme is examined.

Do the coats on vesicles budded from the Golgi apparatus actually cause the budding, or do they simply coat buds (Fig. 1)? One view (the membrane-mediated budding hypothesis) is that budding is an intrinsic property of Golgi membranes not requiring extrinsic coat proteins. Assembly of coats from dispersed subunits is super-imposed upon the intrinsic budding process and is proposed to convert the tips of tubules into vesicles. The alternative view (the coat-mediated budding hypothesis) is that coat formation provides the essential driving force for budding. The membrane-mediated budding hypothesis was inspired by the microtubule-dependent extension of apparently uncoated, 90-nm-diameter membrane tubules from the Golgi apparatus and other organelles in vivo after treatment with brefeldin A, a drug that inhibits the assembly of coat proteins onto Golgi membranes. This hypothesis predicts that tubules will be extended when coat proteins are unavailable to convert tubule-derived membrane into vesicles. Here we use a cell-free system in which coated vesicles are formed from Golgi cisternae to show that, on the contrary, when budding diminishes as a result of immunodepletion of coat protein pools, tubules are not formed at the expense of vesicles. We conclude that coat proteins are required for budding from Golgi membranes.

Human colostrum is an important source of protective, nutritional and developmental factors for the newborn. We have investigated the low abundance proteins in the aqueous phase of human colostrum, after depletion of the major proteins secretory IgA, lactoferrin, alpha-lactalbumin and HSA by immunoabsorption, using 2-D LC and gel-based proteomic methods. One hundred and fifty-one proteins were identified, 83 of which have not been previously reported in human colostrum, or milk. This is the first comprehensive proteomic analysis of human colostrum produced during the first 48 h of lactation.

Abstract. ADP-ribosylation factor (ARF) is a small molecular weight GTP-binding protein (20 kD) and has been implicated in vesicular protein transport. The guanine nucleotide, bound to ARF protein is believed to modulate the activity of ARF but the mechanism of action remains elusive. We have previously reported that ARF binds to Golgi membranes after Brefeldin A-sensitive nucleotide exchange of ARF-bound GDP for GTP),S. Here we report that treatment with phosphatidylcholine liposomes effectively removed 40-60% of ARF bound to Golgi membranes with nonhydrolyzable GTP, presumably by competing for binding of activated ARF to lipid bilayers. This revealed the presence of two different pools of ARF on Golgi membranes. Whereas total ARF binding did not appear to be saturable, the liposome-resistant pool is saturable suggesting that this pool of ARF is stabilized by interaction with a Golgi membrane-component. We propose that activation of ARF by a guanine nucleotideexchange protein results in association of myristoylated ARF.GTP with the lipid bilayer of the Golgi apparatus. Once associated with the membrane, activated ARF can diffuse freely to associate stably with a target protein or possibly can be inactivated by a GTPase activating protein (GAP) activity.