Elizabeth A. Miller

Multiple Cargo Binding Sites on the COPII Subunit Sec24p Ensure Capture of Diverse Membrane Proteins into Transport Vesicles

We have characterized the mechanisms of cargo selection into ER-derived vesicles by the COPII subunit Sec24p. We identified a site on Sec24p that recognizes the v-SNARE Bet1p and show that packaging of a number of cargo molecules is disrupted when mutations are introduced at this site. Surprisingly, cargo proteins affected by these mutations did not share a single common sorting signal, nor were proteins sharing a putative class of signal affected to the same degree. We show that the same site is conserved as a cargo-interaction domain on the Sec24p homolog Lst1p, which only packages a subset of the cargoes recognized by Sec24p. Finally, we identified an additional mutation that defines another cargo binding domain on Sec24p, which specifically interacts with the SNARE Sec22p. Together, our data support a model whereby Sec24p proteins contain multiple independent cargo binding domains that allow for recognition of a diverse set of sorting signals.

Effects of oleate-rich and linoleate-rich diets on the susceptibility of low density lipoprotein to oxidative modification in mildly hypercholesterolemic subjects.

We report the results of feeding oleate- or linoleate-enriched diets for 8 wk to mildly hypercholesterolemic subjects and the resulting alterations in composition and functional properties of their plasma LDL and HDL. LDL isolated from subjects on oleate-enriched diets was less susceptible to copper-mediated oxidation, as measured by conjugated diene and lipid peroxide formation, and less susceptible to LDL-protein modification, as evidenced by reduced LDL macrophage degradation after copper- or endothelial cell-induced oxidation. For all subjects, the percentage of 18:2 in LDL correlated strongly with the extent of conjugated diene formation (r = 0.89, P < 0.01) and macrophage degradation (r = 0.71, P < 0.01). Oxidation of LDL led to initial rapid depletion of unsaturated fatty acids in phospholipids followed by extensive loss of unsaturated fatty acids in cholesteryl esters and triglycerides. Changes in HDL fatty acid composition also occurred. However, HDL from both dietary groups retained its ability to inhibit oxidative modification of LDL. This study demonstrates that alterations in dietary fatty acid composition can effectively alter the fatty acid distribution of LDL and HDL in hypercholesterolemic subjects and that susceptibility to LDL oxidation is altered by these changes. Substitution of monounsaturated (rather than polyunsaturated) fatty acids for saturated fatty acids in the diet might be preferable for the prevention of atherosclerosis.

Antiphospholipid antibodies are directed against epitopes of oxidized phospholipids. Recognition of cardiolipin by monoclonal antibodies to epitopes of oxidized low density lipoprotein.

The optimal clinical management of patients with antiphospholipid antibody syndrome (APS) is uncertain because of a lack of an underlying hypothesis to explain why antiphospholipid autoantibodies (aPL) form to such ubiquitous compounds as phospholipids (PL). In this paper, we demonstrate that many, if not most, aPL are actually directed at neoepitopes of oxidized PL, or neoepitopes generated by adduct formation between breakdown products of oxidized PL and associated proteins. Each cardiolipin (CL) molecule contains four unsaturated fatty acids and is highly susceptible to oxidation, particularly upon exposure to air. Yet, standard anticardiolipin antibodies (aCL) immunoassays routinely bind CL to microtiter wells by evaporation of the ethanol solvent overnight at 4 degrees C. Using a variety of techniques, we demonstrated that rapid oxidation occurs when CL is plated and exposed to air. Sera from apo E-deficient mice, which have high autoantibody titers to oxidized low density lipoprotein, showed a striking time-dependent increase in binding to CL that was exposed to air for increasing periods of time. Monoclonal antibodies to oxidized LDL, cloned from the apo E-deficient mice, also bound to oxidized CL. Both sera and affinity-purified aCL-IgG from APS patients bound to CL progressively as it was oxidized. However, the monoclonal antibodies from apo E-deficient mice, or sera or aCL-IgG from APS patients did not bind to a reduced CL analog that was unable to undergo peroxidation. These data demonstrate that many aPL are directed at neoepitopes of oxidized phospholipids, and suggest that oxidative events may be important in the pathophysiology of APS. In turn, this suggests new therapeutic strategies, possibly including intensive antioxidant therapy.

Cargo selection into COPII vesicles is driven by the Sec24p subunit

Transport of secretory proteins out of the endoplasmic reticulum (ER) is mediated by vesicles generated by the COPII coat complex. In order to understand how cargo molecules are selected by this cytoplasmic coat, we investigated the functional role of the Sec24p homolog, Lst1p. We show that Lst1p can function as a COPII subunit independently of Sec24p on native ER membranes and on synthetic liposomes. However, vesicles generated with Lst1p in the absence of Sec24p are deficient in a distinct subset of cargo molecules, including the SNAREs, Bet1p, Bos1p and Sec22p. Consistent with the absence of any SNAREs, these vesicles are unable to fuse with Golgi membranes. Furthermore, unlike Sec24p, Lst1p fails to bind to Bet1p in vitro, indicating a direct correlation between cargo binding and recruitment into vesicles. Our data suggest that the principle role of Sec24p is to discriminate cargo molecules for incorporation into COPII vesicles.

Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans

Abstract.Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and >200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion.

A cost-benefit analysis of the physical mechanisms of membrane curvature

Many cellular membrane-bound structures exhibit distinct curvature that is driven by the physical properties of their lipid and protein constituents. Here we review how cells manipulate and control this curvature in the context of dynamic events such as vesicle-mediated membrane traffic. Lipids and cargo proteins each contribute energy barriers that must be overcome during vesicle formation. In contrast, protein coats and their associated accessory proteins drive membrane bending using a variety of interdependent physical mechanisms. We survey the energy costs and drivers involved in membrane curvature, and draw a contrast between the stochastic contributions of molecular crowding and the deterministic assembly of protein coats. These basic principles also apply to other cellular examples of membrane bending events, including important disease-related problems such as viral egress.

Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

The secretory pathway is responsible for the synthesis, folding, and delivery of a diverse array of cellular proteins. Secretory protein synthesis begins in the endoplasmic reticulum (ER), which is charged with the tasks of correctly integrating nascent proteins and ensuring correct post-translational modification and folding. Once ready for forward traffic, proteins are captured into ER-derived transport vesicles that form through the action of the COPII coat. COPII-coated vesicles are delivered to the early Golgi via distinct tethering and fusion machineries. Escaped ER residents and other cycling transport machinery components are returned to the ER via COPI-coated vesicles, which undergo similar tethering and fusion reactions. Ultimately, organelle structure, function, and cell homeostasis are maintained by modulating protein and lipid flux through the early secretory pathway. In the last decade, structural and mechanistic studies have added greatly to the strong foundation of yeast genetics on which this field was built. Here we discuss the key players that mediate secretory protein biogenesis and trafficking, highlighting recent advances that have deepened our understanding of the complexity of this conserved and essential process.

ER cargo properties specify a requirement for COPII coat rigidity mediated by Sec13p.

A Fair COP During eukaryotic intracellular membrane traffic, how is membrane curvature imparted by the cytoplasmic proteins that form the COPII coat, which mediates vesicle budding from the endoplasmic reticulum? Čopič et al. (p. 1359, published online 2 February; see the Perspective by Silvius) dissected this process by exploiting yeast bypass-of-sec-thirteen (bst) mutants, which can survive without the otherwise essential COPII coat protein. These bst mutants appear to create a locally altered membrane that is more amenable to deformation by a Sec13-free coat. Membrane curvature of cellular vesicles is generated by altering the symmetry of the cargo and the rigidity of coat proteins. Eukaryotic secretory proteins exit the endoplasmic reticulum (ER) via transport vesicles generated by the essential coat protein complex II (COPII) proteins. The outer coat complex, Sec13-Sec31, forms a scaffold that is thought to enforce curvature. By exploiting yeast bypass-of-sec-thirteen (bst) mutants, where Sec13p is dispensable, we probed the relationship between a compromised COPII coat and the cellular context in which it could still function. Genetic and biochemical analyses suggested that Sec13p was required to generate vesicles from membranes that contained asymmetrically distributed cargoes that were likely to confer opposing curvature. Thus, Sec13p may rigidify the COPII cage and increase its membrane-bending capacity; this function could be bypassed when a bst mutation renders the membrane more deformable.

Vesicle-mediated export from the ER: COPII coat function and regulation

Vesicle trafficking from the endoplasmic reticulum (ER) is a vital cellular process in all eukaryotes responsible for moving secretory cargoes from the ER to the Golgi apparatus. To accomplish this feat, the cell employs a set of conserved cytoplasmic coat proteins - the coat protein II (COPII) complex - that recruit cargo into nascent buds and deform the ER membrane to drive vesicle formation. While our understanding of COPII coat mechanics has developed substantially since its discovery, we have only recently begun to appreciate the factors that regulate this complex and, in turn, ER-to-Golgi trafficking. Here, we describe these factors and their influences on COPII vesicle formation. Properties intrinsic to the GTP cycle of the coat, as well as coat structure, have critical implications for COPII vesicle trafficking. Extrinsic factors in the cytosol can modulate COPII activity through direct interaction with the coat or with scaffolding components, or by changing composition of the ER membrane. Further, lumenal and membrane-bound cargoes and cargo receptors can influence COPII-mediated trafficking in equally profound ways. Together, these factors work in concert to ensure proper cargo movement in this first step of the secretory pathway. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

Regulation of coat assembly--sorting things out at the ER.

The small GTPase Sar1 resides at the core of a regulatory cycle that controls protein export from the ER in COPII vesicles. Recent advances in minimally reconstituted systems indicate continual flux of Sar1 through GTPase cycles facilitates cargo concentration into forming vesicles that ultimately bud from membranes. During export from ER membranes, this GTPase cycle is harnessed through the combinatorial power of multiple coat subunits and cargo adaptors to sort an expanding array of proteins into ER-derived vesicles. The COPII budding machinery is further organized into higher-order structures at transitional zones on the ER surface where the large multi-domain Sec16 protein appears to perform a central function.