Donor Membranes Research Articles

Protein and lipid sorting between the endoplasmic reticulum and the Golgi complex

Vesicular traffic within the early secretory pathway is mediated by COPI– and COPII–coated vesicles. While COPII–coated vesicles appear to be involved exclusively in the export of secretory proteins and lipids from the endoplasmic reticulum (ER), COPI–coated vesicles seem to function in both anterograde and retrograde transport between the ER–Golgi intermediate compartment (IC) and the Golgi as well as in intra–Golgi transport. Here, we focus on (i) the mechanisms how these transport carriers are formed from a given donor membrane; and (ii) the possible mechanisms involved in sorting of proteins and lipids into such transport vesicles.

Steroidogenic Acute Regulatory Protein (StAR) Is A Sterol Transfer Protein

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis by enhancing the delivery of substrate cholesterol from the outer mitochondrial membrane to the cholesterol side chain cleavage enzyme system on the inner membrane. A recombinant StAR protein lacking the first N-terminal 62 amino acid residues that includes the mitochondrial targeting sequence was shown to stimulate the transfer of cholesterol and beta-sitosterol from liposomes to heat-treated mitochondria in a dose-, time-, and temperature-dependent manner. A recombinant mutant StAR protein that cannot stimulate steroidogenesis by isolated mitochondria did not promote sterol transfer. Unlike the more promiscuous lipid transfer protein, sterol carrier protein 2 (SCP2), StAR did not stimulate phosphatidylcholine transfer in our assay system. The recombinant StAR protein increased cholesterol transfer to heat-treated microsomes as well as to heat- and trypsin-treated mitochondria. These observations demonstrate that StAR has sterol transfer activity, which may reflect an ability to enhance desorption of cholesterol from sterol-rich donor membranes. We suggest that the ability of StAR to promote sterol transfer explains its steroidogenic activity.

Biosynthetic protein transport through the early secretory pathway.

Newly synthesized proteins destined for delivery to the cell surface are inserted cotranslationally into the endoplasmic reticulum (ER) and, after their correct folding, are transported out of the ER. During their transport to the cell surface, cargo proteins pass through the various cisternae of the Golgi apparatus and, in the trans-most cisternae of the stack, are sorted into constitutive secretory vesicles that fuse with the plasma membrane. Simultaneously with anterograde protein transport, retrograde protein transport occurs within the Golgi complex as well as from the Golgi back to the ER. Vesicular transport within the early secretory pathway is mediated by two types of non-clathrin coated vesicles: COPI- and COPII-coated vesicles. The formation of these carrier vesicles depends on the recruitment of cytosolic coat proteins that are thought to act as a mechanical device to shape a flattened donor membrane into a spherical vesicle. A general molecular machinery that mediates targeting and fusion of carrier vesicles has been identified as well. Beside a general overview of the various coat structures known today, we will discuss issues specifically related to the biogenesis of COPI-coated vesicles: (1) a possible role of phospholipase D in the formation of COPI-coated vesicles; (2) a functional role of a novel family of transmembrane proteins, the p24 family, in the initiation of COPI assembly; and (3) the direction COPI-coated vesicles may take within the early secretory pathway. Moreover, we will consider two alternative mechanisms of protein transport through the Golgi stack: vesicular transport versus cisternal maturation.

A Putative Heterotrimeric G Protein Inhibits the Fusion of COPI-coated Vesicles: SEGREGATION OF HETEROTRIMERIC G PROTEINS FROM COPI-COATED VESICLES

Heterotrimeric G proteins have been implicated in the regulation of intracellular protein transport, but their mechanism of action remains unclear. In vivo, secretion of chromogranin B, tagged with the green fluorescent protein, was inhibited by the addition of a general activator of trimeric G proteins (AlF4-) to stably transfected Vero cells and resulted in an accumulation of the tagged protein in the Golgi apparatus. In an in vitro assay that reconstitutes intra-Golgi protein transport, we find that a membrane-bound and AlF4--sensitive factor is involved in the fusion reaction. To determine whether this effect is mediated by a heterotrimeric G protein localized to COPI-coated transport vesicles, we determined the presence of G proteins on these vesicles and found that they were segregated relative to the donor membranes. Because G proteins do not have an obvious sorting, retention, or retrieval signal, we considered the possibility that other interactions might be responsible for this segregation. In agreement with this, we found that trimeric G proteins from isolated Golgi membranes were partially insoluble in Triton X-100. Identification of the proteins that interact with the heterotrimeric G proteins in the Golgi-derived detergent-insoluble complex might help to reveal the regulation of protein secretion mediated by heterotrimeric G proteins.

A mitochondrial membrane protein is required for translocation of phosphatidylserine from mitochondria-associated membranes to mitochondria

The mechanism of import of phosphatidylserine (PtdSer) into mitochondria was investigated using a reconstituted system of isolated organelles in vitro in which PtdSer was translocated from donor membranes to mitochondria and was decarboxylated therein. Neither phosphatidylcholine nor phosphatidylethanolamine (PtdEtn) was translocated under the same conditions. Transfer of PtdSer from its site of synthesis on the endoplasmic reticulum and mitochondria-associated membranes [J. E.Vance (1990) J. Biol. Chem. 265, 7248-7256] to its site of decarboxylation on mitochondrial inner membranes is predicted to be mediated by membrane contact. A mitochondrial membrane protein appears to be involved in the translocation event since proteolysis of proteins exposed on the mitochondrial surface potently inhibited PtdSer transfer, whereas proteolysis of surface proteins of mitochondria-associated membranes did not impair the transfer. The nature of the membranes that donate PtdSer to mitochondria in vitro is not crucial since PtdSer of mitochondria-associated membranes, endoplasmic reticulum and microsomes was decarboxylated to PtdEtn with approximately equal efficiency. The translocation of PtdSer to mitochondria was stimulated by magnesium and calcium ions and was inhibited by incubation of mitochondria with sulphydryl group-modifying reagents. Reconstitution of PtdSer translocation/decarboxylation using digitonin-solubilized mitochondria and PtdSer-donor membranes suggested that the putative PtdSer-translocation protein is primarily localized to contract sites between mitochondrial inner and outer membranes. These studies provide evidence for the involvement of a mitochondrial membrane protein in the import of newly-synthesized PtdSer into mitochondria.

Cell-free transport to distinct Golgi cisternae is compartment specific and ARF independent.

The small GTPase ADP-ribosylation factor (ARF) is absolutely required for coatomer vesicle formation on Golgi membranes but not for anterograde transport to the medial-Golgi in a mammalian in vitro transport system. This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism. As one approach to distinguishing between these possibilities, we have characterized two additional cell-free systems that reconstitute transport to the trans-Golgi (trans assay) and trans-Golgi network (TGN assay). Like in vitro transport to the medial-Golgi (medial assay), transport to the trans-Golgi and TGN requires cytosol, ATP, and N-ethylmaleimide-sensitive fusion protein (NSF). However, each assay has its own distinct characteristics of transport. The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event. Depletion of ARF from cytosol abolishes vesicle formation and inhibition by GTPgammaS, but transport in all assays is otherwise unaffected. Purified recombinant myristoylated ARF1 restores inhibition by GTPgammaS, indicating that the GTP-sensitive component in all assays is ARF. We also show that asymmetry in donor and acceptor membrane properties in the medial assay is a unique feature of this assay that is unrelated to the production of vesicles. These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.

Isolation and characterization of full and truncated forms of human breast carcinoma protein BA46 from human milk fat globule membranes.

We have isolated and characterized two proteins of 50 and 30 kDa from human milk fat globule membranes of healthy donors. N-terminal and internal sequencing revealed that the 50-kDa protein is the full-length human breast carcinoma protein BA46 that is highly expressed in human breast tumors. The 30-kDa protein is a truncated form of protein BA46 which consists of the C-terminal factor V/VIII-like domain of BA46 and which appears to anchor BA46 to the milk fat globule membrane. Defective release of the epidermal growth factor domain containing a surface RGD motif may be related to involvement of BA46 in breast cancer.

A molecular basis for retinol stimulation of vesicle budding in vivo and in vitro.

Retinol stimulates the formation of transition vesicles in situ and in all free systems based on rat liver. The stimulation is on vesicle formation from transitional endoplasmic reticulum and not on vesicle fusion with donor membranes. Vesicle budding in the cell free system requires a nucleoside triphosphate and is sensitive to inhibition by thiol reagents. In this report we develop and test a model whereby a retinol-modulated NADH:protein disulfide reductase (NADH oxidase) with protein disulfide-thiol interchange activity is implicated in the vesicle budding mechanism. The protein has the ability to restore activity to scrambled, inactive RNase A and is stimulated or inhibited by retinol depending on the redox environment. Under reducing conditions and in the presence of a chemical reductant such as GSH, the partial reaction stimulated by retinol appears to be the oxidation of membrane thiols. This is the first report of an enzymatic mechanism to explain specific retinol effects both in vivo and in vitro on membrane trafficking not given by retinoic acid.

A Novel Assay Reveals a Role for SolubleN-Ethylmaleimide-sensitive Fusion Attachment Protein in Mannose 6-Phosphate Receptor Transport from Endosomes to the Trans Golgi Network

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (alpha-SNAP) is a soluble protein that enables the NSF ATPase to associate with membranes and facilitate membrane trafficking events. Although NSF and alpha-SNAP have been shown to be required for many membrane transport processes, their role in the transport of mannose 6-phosphate receptors from endosomes to the trans Golgi network was not established. We present here a novel in vitro assay that monitors the transport of cation-dependent mannose 6-phosphate receptors between endosomes and the trans Golgi network. The assay relies on the trans Golgi network localization of tyrosine sulfotransferase and monitors transport of mannose 6-phosphate receptors engineered to contain a consensus sequence for modification by this enzyme. Using this new assay we show that alpha-SNAP strongly stimulates transport in reactions containing limiting amounts of cytosol. Together with alpha-SNAP, NSF can increase the extent of transport. These data show that alpha-SNAP, a soluble component of the SNAP receptor machinery, facilitates transport from endosomes to the trans Golgi network.

Biogenesis of COPI-coated transport vesicles

Biosynthetic protein transport and sorting along the secretory pathway represents the last step in biosynthesis of a variety of proteins. Proteins destined for delivery to the cell surface are inserted cotranslationally into the endoplasmic reticulum (ER) and, after their correct folding, are transported out of the ER towards their final destinations. The successive compartments of the secretory pathway are connected by vesicular shuttles that mediate delivery of cargo. The formation of these carrier vesicles depends on the recruitment of cytosolic coat proteins that are thought to act as a mechanical device to shape a flattened donor membrane into a spherical vesicle. A general molecular machinery that mediates targeting and fusion of carrier vesicles has also been identified. This review is focused on COPI-coated vesicles that operate in protein transport within the early secretory pathway. Rather than representing a general overview of the role of COPI-coated vesicles, this mini-review will discuss mechanisms specifically related to the biogenesis of COPI-coated vesicles: (i) a possible role of phospholipase D in the formation of COPI-coated vesicles, (ii) a functional role of a novel family of transmembrane proteins, the p24 family, in the initiation of COPI assembly, and (iii) the direction COPI-coated vesicles may take within the early secretory pathway.

ADP ribosylation factor 1 is required for synaptic vesicle budding in PC12 cells.

Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein-T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041-1049). A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

Is dynamin really a ‘pinchase’?

The motivation for this article was a recent conversation with an author of a major cell-biology textbook who was gratified that the problem of ?pinching off? membrane vesicles from donor membranes had been solved.It was now known, the author claimed, that the large GTPase dynamin was a ?pinchase? severing the necks of budding vesicles.We are concerned that this appealing speculation has been prematurely elevated to cell-biological dogma.Furthermore, by seeing dynamin exclusively as a pinchase we might fail to recognize alternative functions.In this article, we review briefly the evidence that dynamin is a pinchase, discuss some problems with that speculation and show that other possible functions for dynamin may be at least as appealing.

Modelling intracellular fatty acid transport: possible mechanistic role of cytoplasmic fatty acid-binding protein

A computer model is presented in which the role of cytoplasmic fatty acid-binding protein (FABP) in the intracellular translocation of fatty acids (FA) from one membrane to an opposite membrane is studied. The model consists of a cubical space, in which FABP and FA are allowed to diffuse at random. The amount of FA released from the donor membrane and reaching an opposite acceptor membrane is calculated in a variety of conditions. The data provided by the various simulations suggest that FABP can play a significant role in intracellular FA transport only if FABP is able to take up FA directly from FA containing membranes and to directly deliver FA to an acceptor membrane, thus preventing the unfavourable thermodynamical situation in which FA must solubilize in an aqueous environment prior to binding to FABP.

Regulatory interactions in the recognition of endocytic sorting signals by AP-2 complexes.

Many plasma membrane proteins destined for endocytosis are concentrated into clathrin-coated pits through the recognition of a tyrosine-based motif in their cytosolic domains by an adaptor (AP-2) complex. The mu2 subunit of isolated AP-2 complexes binds specifically, but rather weakly, to proteins bearing the tyrosine-based signal. We now demonstrate, using peptides with a photoreactive probe, that this binding is strengthened significantly when the AP-2 complex is present in clathrin coats, indicating that there is cooperativity between receptor-AP-2 interactions and coat formation. Phosphoinositides with a phosphate at the D-3 position of the inositol ring, but not other isomers, also increase the affinity of the AP-2 complex for the tyrosine-based motif. AP-2 is the first protein known (in any context) to interact with phosphatidylinositol 3-phosphate. Our findings indicate that receptor recruitment can be coupled to clathrin coat assembly and suggest a mechanism for regulation of membrane traffic by lipid products of phosphoinositide 3-kinases.

Direct and GTP-dependent interaction of ADP ribosylation factor 1 with coatomer subunit beta.

A site-directed photocrosslink approach was used to elucidate components that interact directly with ADP- ribosylation factor (ARF)-GTP during coat assembly. Two ARF mutants were generated that contain a photolabile amino acid at positions distant to each other within the ARF molecule. Here we show that one of the two positions specifically interacts with coatomer subunit beta both on Golgi membranes and in isolated coat protein complex type I (COPI)-coated vesicles. Thus, a direct and GTP-dependent interaction of coatomer via beta-coat protein complex (COP) with ARF is involved in the coating of COPI-coated vesicles. These data implicate a bivalent interaction of the complex with the donor membrane during vesicle formation.

A comparative study of structural transitions in erythrocyte membranes of adult donors and neonates

Differential scanning microcalorimetry and electron microscopy using freeze-fracture technique shows that the erythrocyte plasma membrane of adults differs in several parameters of heat absorption from that of neonates. In particular, electron microscopy demonstrates that the number and diameter of intramembrane particles are smaller in neonatal erythrocyte membranes. The results of this study account for a number of features possessed by neonatal erythrocytes.

ATP-dependent formation of free synaptic vesicles from PC12 membranes in vitro.

Synaptic vesicles are released from membranes during incubation at 37 degrees C in the presence of ATP (adenosine triphosphate). The donor membranes are a rapidly sedimenting fraction derived from the neuroendocrine cell line PC12 (pheochromocytoma 12). These starting membranes contain the synaptic vesicle proteins, synaptophysin and SV2, and the endosomal markers transferrin receptor and cation-independent MPR (mannose 6-phosphate receptor). Incubating the membranes in vitro increased the amount of organelles that migrate as synaptic vesicles in velocity sedimentation gradients. The synaptic vesicle fractions that contain both synaptophysin and SV2 do not contain endosomal markers. A synaptic vesicle increase in vitro is time-, cytosol-, ATP- and temperature-dependent and is inhibited by NEM (N-ethylmaleimide), BFA (brefeldin A) and aluminum fluoride, but not GTP gamma S (guanosine-5'O-C3-thiotriphosphate). The production of synaptic vesicles under these conditions is unlike the de novo generation of vesicles from endosomes (1). Incubation in vitro under the conditions described here may allow the final stages of synaptic vesicle formation, uncoating or undocking, to occur but not the initiation of formation de novo.

The production of post-Golgi vesicles requires a protein kinase C-like molecule, but not its phosphorylating activity.

We have recently described a system that recreates in vitro the generation of post-Golgi vesicles from purified Golgi fractions obtained from virus-infected MDCK cells in which the vesicular stomatitis virus-G envelope glycoprotein had been allowed to accumulate in vivo in the TGN. Vesicle formation, monitored by the release of the viral glycoprotein, was shown to require the activation of a GTP-binding ADP ribosylation factor (ARF) protein that promotes the assembly of a vesicle coat in the TGN, and to be regulated by a Golgi-associated protein kinase C (PKC)-like activity. We have now been able to dissect the process of post-Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and another of vesicle scission, neither of which requires an ATP supply. The first stage can occur at 20 degrees C, and includes the GTP-dependent activation of the ARF protein, which can be effected by the nonhydrolyzable nucleotide analogue GTP gamma S, whereas the second stage is nucleotide independent and can only occur at a higher temperature of incubation. Cytosolic proteins are required for the vesicle scission step and they cannot be replaced by palmitoyl CoA, which is known to promote, by itself, scission of the coatomer-coated vesicles that mediate intra-Golgi transport. We have found that PKC inhibitors prevented vesicle generation, even when this was sustained by GTP gamma S and ATP levels reduced far below the K(m) of PKC. The inhibitors suppressed vesicle scission without preventing coat assembly, yet to exert their effect, they had to be added before coat assembly took place. This indicates that a target of the putative PKC is activated during the bud assembly stage of vesicle formation, but only acts during the phase of vesicle release. The behavior of the PKC target during vesicle formation resembles that of phospholipase D (PLD), a Golgi-associated enzyme that has been shown to be activated by PKC, even in the absence of the latter's phosphorylating activity. We therefore propose that during coat assembly, PKC activates a PLD that, during the incubation at 37 degrees C, promotes vesicle scission by remodeling the phospholipid bilayer and severing connections between the vesicles and the donor membrane.

Determination of diprivan in urine by a supported liquid membrane technique and liquid chromatography-electrochemical detection.

A supported liquid membrane technique was used for the extraction and enrichment of propofol in a spiked sample of urine. An acidic solution of propofol and thymol as an internal standard was passed over the membrane and after enrichment the acceptor solution was analyzed by LC with an electrochemical detector. The acceptor and donor pH, flow-rate, and volume of donor and different membrane solvents were varied to optimize the extraction efficiency. The detection limit for 100 ml of a spiked urine sample was 10 ppt of propofol.

Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein.

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.