Membrane deformation and separation

Biological membranes are highly dynamic (e.g., during cell division, organelle biosynthesis, vesicular transport, and neurotransmitter release). They can be shaped into protein-coated transport vesicles or tubules and undergo regulated fusion. The life of transport vesicles depends on highly specific and tightly regulated protein machineries, which not only shape the donor membrane into nascent budding structures but also help to overcome the energy barrier to break the bilayers apart in order to pinch off nascent vesicles. Ultimately, vesicular membranes have to fuse with a target lipid bilayer, a process that again requires remodeling. Here, we highlight recent insights into mechanisms that lead to membrane deformation in the process of vesicular budding

Lactosylceramide is synthesized in the lumen of the Golgi apparatus

AbstractRecently, synthesis of lactosylceramide has been described to occur on the cytosolic face of the Golgi [(1991) J. Biol. Chem. 266, 20907-20912]. The reactions following in the biosynthesis of higher glycosphingolipids are known to take place in the lumen of the Golgi. For our understanding of the functional organization of the multiglycosyltransferase system of glycosphingolipid synthesis in the Golgi, the knowledge of the topology of individual reactions is a prerequisite. We have developed a simple and quick assay system for sphingolipid biosynthesis and have obtained evidence that lactosylceramide is synthesized in the lumen of the Golgi. Because lactosylceramide is generated by galactosylation of glucosylceramide which, in turn, is synthesized from ceramide and UDP-Glc on the cytosolic surface of the Golgi apparatus, further efforts will be directed to the characterization of a glucosylceramide-translocator in the Golgi membranes rather than a lactosylceramide-translocator

Supplemental Materials: Novel Isotypic g/z-Subunits Reveal Three Coatomer Complexes in Mammals

Fig. A 1 shows a Western blot analyzing the specificity of the antibodies against g- and z-isotypes. These antibodies were probed in different mammalian species (Fig. A 2). Coatomer was labeled with [35S]-methionine for different time periods, and the incorporation of [35S]-methionine as well as the corresponding stoichiometries of the coatomer subunits are shown in Fig. A 3. Characterization of anti-coatomer antibody 883 is depicted in Fig. A 4

Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

BACKGROUND: The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. RESULTS: Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. CONCLUSION: Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo

Dual roles of the transmembrane protein p23/TMP21 in the modulation of amyloid precursor protein metabolism

BACKGROUND: Alzheimer's disease (AD) is characterized by cerebral deposition of β-amyloid (Aβ) peptides. Aβ is released from ectodomain cleaved amyloid precursor protein (APP) via intramembranous proteolysis by γ-secretase, a complex consisting of presenilin and a few other proteins. p23/TMP21, a member of the p24 family type I transmembrane proteins, was recently identified as a presenilin complex component capable of modulating γ-secretase cleavage. The p24 family proteins form oligomeric complexes and regulate vesicular trafficking in the early secretory pathway, but their role in APP trafficking has not been investigated. RESULTS: Here, we report that siRNA-mediated depletion of p23 in N2a neuroblastoma and HeLa cells produces concomitant knockdown of additional p24 family proteins and increases secretion of sAPP. Furthermore, intact cell and cell-free Aβ production increases following p23 knockdown, similar to data reported earlier using HEK293 cells. However, we find that p23 is not present in mature γ-secretase complexes isolated using an active-site γ-secretase inhibitor. Depletion of p23 and expression of a familial AD-linked PS1 mutant have additive effects on Aβ(42 )production. Knockdown of p23 expression confers biosynthetic stability to nascent APP, allowing its efficient maturation and surface accumulation. Moreover, immunoisolation analyses show decrease in co-residence of APP and the APP adaptor Mint3. Thus, multiple lines of evidence indicate that p23 function influences APP trafficking and sAPP release independent of its reported role in γ-secretase modulation. CONCLUSION: These data assign significance to p24 family proteins in regulating APP trafficking in the continuum of bidirectional transport between the ER and Golgi, and ascribe new relevance to the regulation of early trafficking in AD pathogenesis

Journeys through the Golgi—taking stock in a new era

The Golgi apparatus is essential for protein sorting and transport. Many researchers have long been fascinated with the form and function of this organelle. Yet, despite decades of scrutiny, the mechanisms by which proteins are transported across the Golgi remain controversial. At a recent meeting, many prominent Golgi researchers assembled to critically evaluate the core issues in the field. This report presents the outcome of their discussions and highlights the key open questions that will help guide the field into a new era

GORAB scaffolds COPI at the trans-Golgi for efficient enzyme recycling and correct protein glycosylation

COPI is a key mediator of protein trafficking within the secretory pathway. COPI is recruited to the membrane primarily through binding to Arf GTPases, upon which it undergoes assembly to form coated transport intermediates responsible for trafficking numerous proteins, including Golgi-resident enzymes. Here, we identify GORAB, the protein mutated in the skin and bone disorder gerodermia osteodysplastica, as a component of the COPI machinery. GORAB forms stable domains at the trans-Golgi that, via interactions with the COPI-binding protein Scyl1, promote COPI recruitment to these domains. Pathogenic GORAB mutations perturb Scyl1 binding or GORAB assembly into domains, indicating the importance of these interactions. Loss of GORAB causes impairment of COPI-mediated retrieval of trans-Golgi enzymes, resulting in a deficit in glycosylation of secretory cargo proteins. Our results therefore identify GORAB as a COPI scaffolding factor, and support the view that defective protein glycosylation is a major disease mechanism in gerodermia osteodysplastica.Peer reviewe