Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chaudhuri, S.
Right arrow Articles by Berger, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chaudhuri, S.
Right arrow Articles by Berger, M.
(Journal of Leukocyte Biology. 2001;70:669-676.)
© 2001 by Society for Leukocyte Biology

Association of ARF and Rabs with complement receptor Type-1 storage vesicles in human neutrophils

Subhendu Chaudhuri*, Anoopa Kumar2 and Melvin Berger*

* Department of Pediatrics, Case Western Reserve University School of Medicine, and
{dagger} Division of Nephrology, Veterans Administration Hospital, Cleveland, Ohio

Correspondence: Melvin Berger, M.D., Ph.D., Division of Pediatric Immunology, Rainbow Babies’ and Children’s Hospital, Room 594, 11100 Euclid Ave., Cleveland, OH 44106. E-mail: mxb12{at}po.cwru.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During neutrophil activation, the properties of the cell are rapidly altered by increases in the surface expression of functionally important receptors and adherence molecules. At the same time, endocytic and phagocytic activities increase. These alterations require precise regulation of membrane and protein movement, which is achieved, at least in part, by bidirectional movement of small transport vesicles. GTP-binding proteins, including Rabs and ADP-ribosylation factors (ARFs), play critical roles in regulating vesicle trafficking in other types of cells. The ability to immunoisolate the "secretory" vesicle subpopulation in which complement receptor type 1 (CR1) is stored allowed us to determine which types of low-molecular-weight GTP-binding proteins interact with these vesicles and under what conditions. CR1-containing vesicles from resting human neutrophils constitutively copurify with Rabs 3a, 4, and 5a, and reversibly bind an ARF, likely ARF1. ARF binding is dependent on free Mg2+ and is enhanced by GTP{gamma}S. Mg2+ at 0.4 µM is necessary for half-maximal binding of ARFs to CR1 storage vesicles. Artificial phospholipid vesicles and primary and secondary granules from human neutrophils do not bind ARFs themselves and do not compete for recruitment of ARFs to CR1 vesicles, suggesting that specific membrane environments and/or proteins on these vesicles stabilize the ARF-GTP-Mg2+ complex. Free Ca2+ at 300 nM does not inhibit ARF binding to CR1 storage vesicles, but 10 mM Ca2+ does reduce such binding. These findings suggest that ARF-GTP specifically and reversibly interacts with CR1 storage vesicles in human polymorphonuclear leukocytes and may play a role in regulating their transport.

Key Words: low-molecular-weight GTP-binding proteins • transport vesicles • phagocytosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human neutrophils contain heterogeneous populations of granules and vesicles whose contents have diverse functions and which appear at different stages of cell development [1 ]. In addition to secretory proteins, these subcellular compartments serve as storage pools of readily mobilizable membrane proteins which translocate to the plasma membrane during cell activation, allowing the cell to respond quickly to external stimuli. At the same time, endocytic vesicles are internalized to partially balance the membrane flow. Electron microscopic and subcellular fractionation studies of resting neutrophils show that a large intracellular pool of complement receptor type 1 (CR1) is localized in a distinct type of "secretory vesicles," which are well separated from primary and secondary granules on density gradients [1 2 3 ]. Development of a method of purification of CR1 storage vesicles on immunobeads using antibodies against the cytoplasmic tail of CR1 has confirmed that CR1 is stored in a subset of secretory vesicles [4 ]. Moreover, this purification technique has demonstrated that the CR1 storage vesicles also contain diverse types of membrane and soluble proteins, consistent with the hypothesis that they are formed by reinternalization of membrane proteins, which accompanies endocytosis of soluble proteins [1 ]. Secretory vesicles are highly mobile and are readily translocated to the plasma membrane upon cell activation, with more rapid kinetics than secretion of traditional granule contents and in response to lower concentrations of agonists such as N-formyl-methionyl-leucyl-phenylalanine [1 2 3 4 5 ]. Low-molecular-weight guanosine triphosphate (GTP)-binding proteins are frequently involved in vesicular trafficking in other types of cells, so we postulated that they might also be involved in translocation of the CR1 storage vesicles in polymorphonuclear leukocytes (PMN). The availability of a method for immunoisolation of CR1 vesicles, therefore, enabled us to study the interactions of these vesicles with low-molecular-weight GTP-binding proteins in vitro, without a requirement for other separation techniques which would give more heterogeneous vesicle mixtures.

Low-molecular-weight monomeric GTP-binding proteins including adenosine diphosphate-ribosylation factors (ARFs) and Rabs are members of the Ras superfamily [6 7 8 9 10 11 12 13 14 ]. Thirty Rabs [6 ] and six ARFs [15 ] have been identified so far in mammalian cells. The common feature of these GTP-binding proteins is their ability to shuttle between a soluble guanosine diphosphate (GDP)-bound form and a membrane-associated GTP-bound state as they regulate vesicular trafficking. Some Rabs are expressed universally in cells that share common transport pathways, and others are found only in highly differentiated cells with unique transport pathways [16 , 17 ]. We observed that Rabs 3a, 4, and 5a were constitutively present in the immunoisolated CR1 vesicles, whereas the association of ARF with these vesicles was variable and could be increased by incubation with cytosol, suggesting regulatable binding. Of six known mammalian ARF proteins with molecular masses of ~21 kDa, ARF1 is the best characterized. It participates in a wide range of functions, including regulation of vesicle traffic along both exocytic and endocytic pathways, maintenance of organelle structure, and assembly of coat proteins, and it may also be a cofactor for cholera toxins [7 8 9 ]. In this study we present evidence for the recruitment of an ARF from the cytosolic fraction of neutrophils to isolated CR1 vesicles in the presence of GTP{gamma}S, and the requirements for Mg2+ versus Ca2+ in this binding.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of CR1 storage vesicles and cytosol from nitrogen cavitates of resting PMN
Heparinized peripheral blood was obtained from normal donors who were afebrile, free of known infection, and not taking any medications, and PMN were isolated as described previously [1 , 4 ]. The isolated PMN were then suspended in relaxation buffer [100 mM KCl, 3 mM NaCl, 1 mM ATP(Na2), 3.5 mM MgCl2, 10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) (pH 7.3)] containing protease inhibitors and were subjected to nitrogen cavitation at 4°C for the preparation of PMN lysates [1 , 3 , 4 ]. For the isolation of CR1 storage vesicles from these PMN lysates, the cavitate was initially separated on preliminary 2-layer Percoll gradients to obtain cytosol, a light membrane fraction also termed the "{gamma} band," and a mixture of primary and secondary granules [3 ]. Cytosol and the {gamma} band (5–6 mL) were freed from Percoll by centrifugation without a cushion of Percoll according to a procedure described previously [4 ]. Cytosol was stored at -70°C until further use. CR1 storage vesicles were subsequently immunoisolated from the Percoll-free {gamma} band by using a magnetic bead system (Dynabeads, M280; Dynal, Inc., Lake Success, NY) with rabbit antibodies against the cytoplasmic tail of CR1 and sheep anti-rabbit antibodies on the beads, exactly as described previously [4 ]. Vesicles purified in this way are free from contamination, as judged by the absence of marker enzymes of primary and secondary granules [4 ]. In addition, Golgi proteins such as p230 [18 ], golgin-84 [19 ], and GS28 [20 ] were absent from the immunoisolated CR1 vesicles (data not shown).

Assay for myeloperoxidase and preparation of primary- and secondary-granule mixture
Myeloperoxidase activity was determined with 0.006% hydrogen peroxide, 2.5 mM 4-aminoantipyrine, and 16 mg/mL of phenol in 100 mM Tris-HCl buffer (pH 8.5) in 96-well microtiter plates. The change in absorbance was monitored continuously at 490 nm at room temperature for 2–3 min.

A mixture of primary and secondary granules was prepared on 2-layer Percoll gradients from nitrogen cavitates of PMN as described above. One-milliliter fractions were collected after removal of cytosol and the {gamma} band from the top of the gradient. Fractions with myeloperoxidase activity were pooled and freed from Percoll by centrifugation at 180,000 g for 1 h on a cushion of 0.5 mL of undiluted Percoll according to the published procedure [1 , 4 ]. This mixture of primary and secondary granules, which was positive for myeloperoxidase, was kept at 4°C until further use.

Association of low-molecular-weight GTP-binding proteins with immunoisolated CR1 storage vesicles
As a first step in studying the interaction of small GTP-binding proteins with CR1 vesicles, we checked for the presence of Rabs and ARFs in isolated CR1 vesicles, cytosol, and the mixture of primary and secondary granules by Western blotting as described below.

To determine if additional Rabs and ARF could be recruited from cytosol to immunoisolated CR1 vesicles, we performed incubations in the presence and absence of cytosol at 37°C for 30 min in relaxation buffer [100 mM KCl, 3 mM NaCl, 1 mM ATP, 3.5 mM MgCl2, 10 mM PIPES (pH 7.3)] containing protease inhibitors [4 ] and appropriate guanine nucleotides. Reactions were terminated with 4 volumes of ice-cold phosphate-buffered saline (PBS) containing protease inhibitors. In initial experiments, the beads were harvested with a magnet, washed five times with PBS buffer containing protease inhibitors, and prepared for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In later experiments, 3.5 mM Mg2+ was included in the wash buffer. Additional experiments with varying amounts of cytosol, different nucleotides, and different concentrations of cations (see below) were also performed using this protocol. Proteins attached to immunoisolated CR1 vesicles on the magnetic beads were eluted by suspension in 3 volumes of nonreducing SDS sample buffer and boiling for 2 min. Proteins were then separated by SDS-PAGE [21 ] and detected by Western blotting.

Similar experiments were performed to determine if primary and secondary granules could also recruit ARF proteins from cytosol on incubation at 37°C for 30 min in relaxation buffer as above. Granules from the reaction mixture were separated on modified 2-layer Percoll gradients containing 3 mL of 1.065-g/mL Percoll with a cushion of 0.5 mL of 1.12-g/mL Percoll. Fractions (0.5 mL) were assayed for myeloperoxidase activity as described above. Myeloperoxidase-positive fractions were pooled and freed from Percoll, and the recovered granules were concentrated to 50 µL on microconcentrators (Microcon 10; Amicon, Beverly, MA) before being subjected to SDS-PAGE for analysis of ARF binding by Western blotting.

Magnesium-dependent binding of ARF to immunoisolated CR1 vesicles
To determine of the effect of Mg2+ on the binding of ARF to CR1 storage vesicles, Mg2+ was omitted from the relaxation buffer before nitrogen cavitation. No attempt was made to remove intracellular Mg2+ or nucleotides from cytosol. Mg2+-EDTA buffer was made with varying concentrations of Mg2+ in relaxation buffer in the presence of 5 mM EDTA. Mg2+-EGTA buffer was made likewise. The concentrations of free Mg2+ at 37°C for both buffers (ionic strength = 0.109) were calculated as described by Portezehl et al. [21 ].

Effects of granules and phospholipid vesicles on ARF recruitment to immunoisolated CR1 vesicles
In some experiments we included granules or artificial phospholipid vesicles in the reaction mixture with cytosol and CR1 vesicles on beads to determine if the presence of these other types of lipid membranes would influence the recruitment of ARF to CR1 storage vesicles. A mixture of primary and secondary granules containing myeloperoxidase activity was isolated from PMN cavitate on 2-layer Percoll gradients as described above and used for these experiments to determine if they would compete with the CR1 vesicles for binding of ARF.

To provide further assurance that the observed binding of ARF was not just a hydrophobic effect, we also used phospholipid vesicles containing phosphatidylethanolamine, phosphatidylinositol 4,5-diphosphate, and dipalmitoyl phosphotidylcholine in a molar ratio of 10:1.5:1. This composition was selected to allow determination of activation of phospholipase D (PLD) upon binding of ARF, as has been reported by others [10 , 14 , 23 ]. PLD activity in these vesicles would result in liberation of choline, which would have been detected by chemiluminiscence of luminol in the presence of horseradish peroxidase by H2O2 from the oxidation of liberated choline by choline oxidase. The chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), and phospholipid vesicles were prepared according to the work of Brown et al. [14 ]. Phospholipid vesicles (56 and 168 µL) were incubated for 30 min at 37°C with CR1 vesicles and cytosol in a final volume of 1 mL of incubation buffer containing 50 mM HEPES buffer (pH 7.5) with 3 mM EGTA, 80 mM KCl, 3 mM MgCl2, and 2 mM CaCl2, with or without 400 µM GTP{gamma}S. After incubation, CR1 vesicles were isolated with a magnet, washed as before, and prepared for SDS-PAGE. Incubations without granules or phospholipids were run in parallel as controls for the amount of ARF bound to CR1 vesicles alone.

Immunoblotting
Protein samples were analyzed on SDS-PAGE gels (12 or 15% polyacrylamide) under nonreducing conditions in a Mini Protean II Dual Slab Cell (Bio-Rad, Hercules, CA) using prestained low-molecular-weight markers from Bio-Rad. Separated proteins were transferred onto nitrocellulose membranes (Pharmacia, Uppsala, Sweden) using 192 mM glycine in 25 mM Tris (pH 8.3) containing 20% methanol [23 ]. The membrane was blocked with 5% nonfat dry milk and 1% bovine serum albumin in PBS with 0.05% Tween 20 (Fisher Scientific, Pittsburgh, PA) for 1 h at room temperature and was subsequently washed four times with PBS buffer containing 0.05% Tween 20. The membrane was then probed with primary antibody for 1 h at room temperature. Polyclonal rabbit antibody to human ARF1, as well as recombinant human ARF1 (used as a positive control) were kind gifts of Dr. Sylvain Bourgoin of Centre de Recherche en Rhumatologie et Immunologie, Quebec, Canada. Isoform-specific polyclonal and monoclonal antibodies to human Rabs were purchased from Santa Cruz Biotechnology, Inc. and BD Transduction Laboratories (San Diego, CA), respectively. Monoclonal antibodies to Golgi proteins were also obtained from Transduction Laboratories. Control proteins were always run in parallel to identify Rabs and ARF. Horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (Amersham, Little Chalfont, Buckinghamshire, UK) and anti-mouse immunoglobulin G (BD Transduction Laboratories) were used as secondary antibodies to probe the membrane for 1 h, and bound antibody was detected by enhanced chemiluminescence using a kit from Amersham. In some cases we used alkaline phosphatase-conjugated secondary antibody.

Estimation of protein
Bradford reagent from Bio-Rad was used for the estimation of proteins in a 96-well microtiter plate using a Thermomax reader (Molecular Devices, Sunnyvale, CA) according to the manufacturer’s protocol.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Copurification of Rab proteins with immunoisolated CR1 storage vesicles
We started with the premise that most membrane-bound organelles in secretory and endocytic pathways bind on their cytosolic faces a defined set of Rab proteins. To test this hypothesis, we screened immunoisolated CR1 storage vesicles and subcellular fractions of PMN for Rab proteins with a panel of commercially available antibodies against Rabs. In this study we were able to detect Rabs 3a, 4, and 5a on isolated CR1 storage vesicles (Fig. 1 , lane 5). Incubation with cytosol with or without GTP{gamma}S resulted in no significant increase in the amount of Rab proteins bound to vesicles (Fig. 1 , lane 5 vs. lanes 6 and 7). Rabs 1, 2, 7, 9, and 11 were absent from CR1 storage vesicles. Occasionally, small amounts of Rab6 were also found in association with these vesicles isolated on magnetic beads, with or without the addition of cytosol (data not shown). Rabs 3a, 4, and 5a were also present in the mixture of primary and secondary granules (Fig. 1 , lane 4). In addition, Rabs 1, 2, 6, 7, 9, and 11 were also detectable in the mixture of primary and secondary granules (data not shown), and all except Rab1a were present in cytosol (data not shown). We thus conclude that Rabs 3a, 4, and 5a are constitutively associated with this subpopulation of vesicles and that Rabs 2, 7, 9, and 11, which are associated with primary and secondary granules, are absent from the CR1 storage vesicles.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 1. Detection of Rabs 3a, 4, and 5a on immunoisolated CR1 storage vesicles by Western blotting. Incubations were carried out at 37°C for 30 min in 10 mM PIPES, pH 7.3, containing 3.5 mM Mg2+, 1 mM ATP, and protease inhibitors. Lane 1, control; lane 2, PMN lysate; lane 3, cytosol; lane 4, mixture of primary and secondary granules; lane 5, CR1 vesicles alone; lane 6, CR1 vesicles recovered after incubation with cytosol; lane 7, similar to lane 6, but with GTP{gamma}S in the incubation mixture.

 
Recruitment of ARF from cytosol to immunoisolated CR1 vesicles derived from PMN
In preliminary studies, we found variable amounts of ARF in different preparations of immunoisolated CR1 vesicles. We postulated that this might be due to the reversible association of one or more members of this family of GTP-binding proteins with the vesicles, because ARF proteins are predominantly cytosolic, as shown in Figure 2B , lanes 2 and 3. We never observed any ARF protein in the mixture of primary and secondary granules using this antibody, which recognizes multiple isoforms of ARF GTPases (see below). We therefore studied the conditions under which isolated CR1 storage vesicles could recruit ARF from cytosol. The amount of ARF bound to the isolated vesicles was small but usually visible (Fig. 2A , lane 2). Incubation with cytosol markedly increased the amount of ARF remaining with the CR1 vesicles bound to the magnetic beads (Fig. 2A , lane 3). We found that recruitment of ARF from the cytosol to isolated CR1 storage vesicles was further enhanced in the presence of GTP{gamma}S (Fig. 2A , lane 4). In contrast to the CR1 storage vesicles, ARF was not present in the granule fraction (Fig. 2B , lane 4, and Fig. 2A , lane 5) and could not be recruited to granules by incubation with cytosol in the presence of GTP{gamma}S (Fig. 2B , lane 5) under the same conditions that promoted its association with CR1 vesicles. These results therefore suggest that the variable binding we observed in early experiments was due to variable association of cytosolic ARF with the CR1 vesicles and not to contamination of the CR1 vesicles with granule membranes.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 2. ARF is recruited to CR1 vesicles from the cytosol, but not from granules. Reaction conditions were the same as those for Fig. 1 . ARF was detected by enhanced chemiluminescence on Western blots as described in Materials and Methods. (A) Interaction of CR1 vesicles with and without cytosol. Proteins were isolated by SDS–12% PAGE and transferred to a nitrocellulose membrane for detection of ARF. Lane 1, ARF control; lane 2, CR1 vesicles alone; lane 3, CR1 vesicles recovered after incubation with cytosol; lane 4, similar to lane 3, with 400 µM GTP{gamma}S in the incubation mixture; lane 5, CR1 vesicles with granules and 400 µM GTP{gamma}S, without cytosol. (B) Interaction of granules with cytosol. Proteins isolated on an SDS–15% PAGE gel were transferred to a nitrocellulose membrane, which was probed for ARF by Western blotting. Lane 1, ARF control; lanes 2 and 3, 3 and 9 µg of cytosol, respectively; lane 4, 1.8 µg of the mixture of primary and secondary granules; lane 5, recovered granules after incubation with cytosol in the presence of GTP{gamma}S.

 
Taken together, these results suggest that, in resting cells, ARF is mostly present as a soluble protein in cytosol [23 ], but they show that, unlike primary and secondary granules, CR1 storage vesicles have a unique ability to recruit ARF from the cytosol, which is increased by GTP{gamma}S. Brefeldin A (10 µg/mL) did not inhibit ARF recruitment to CR1 storage vesicles (data not shown), and we have previously found that brefeldin treatment of intact PMN did not alter the net up-regulation of CR1 on the plasma membrane in response to chemoattractants (unpublished data). We speculate that this may be due to the presence of brefeldin A-resistant guanine nucleotide exchange proteins such as cytohesin 1 and ARNO [24 ]. Figure 3 shows that increasing amounts of ARF are recruited to CR1 vesicles with increasing amounts of cytosol. In this experiment, the bound ARF was stabilized by inclusion of 3.5 mmol/L of MgCl2 in the buffer used for washing the CR1 vesicle-bearing beads after their incubation with or without cytosol.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Effect of increasing concentrations of cytosolic proteins on ARF binding to CR1 vesicles. ARF was recruited from the cytosol to immunoisolated CR1 vesicles under experimental conditions similar to those described for Figure 2 , but the beads were washed with a buffer containing 3.5 mM MgCl2 after the incubation with or without cytosol. Proteins were separated on an SDS–12% PAGE gel, and ARF was detected by enhanced chemiluminescence. Lane 1, ARF control; lane 2, CR1 vesicles incubated without cytosol, and without GTP{gamma}S; lanes 3–8, CR1 vesicles incubated with 10, 50, 75, 100, 125, and 150 µg of cytosol protein, respectively, each with 400 µM GTP{gamma}S.

 
Effects of nucleotides and divalent cations on ARF binding to CR1 vesicles
Since binding of proteins in these families to membranes is generally GTP dependent and also is influenced by cations, we studied the effects of nucleotides, cations, and chelators. Figure 4 shows that while addition of GDP did not promote binding of ARF from cytosol to CR1 vesicles (lane 4), the addition of GTP{gamma}S enhanced ARF binding (lane 5). The amount of ARF bound to CR1 storage vesicles does not change from 1 to 400 µM GTP{gamma}S (unpublished observation). The effect of 200 µM GTP{gamma}S in recruiting ARF to CR1 storage vesicles was increased further in the presence of 10 mM EDTA (lane 6). Although the increase in expression of CR1 on the plasma membrane following stimulation of PMN with agonists such as N-formyl-methionyl-leucyl-phenylalanine is Ca2+ dependent, we found that addition of excess Ca2+ (10 mM) in the incubation buffer resulted in decreased binding of ARF to the CR1 vesicles in these studies (Fig. 4 , lane 7). However, there was no inhibition at other concentrations tested, and most experiments were carried out with 300 nM free Ca2+ (see Fig. 7 below), a concentration frequently employed in assays of PLD activity [14 ]. We speculate that the requirement for Ca2+ in up-regulation of CR1 expression on the plasma membrane during PMN activation is due to involvement in a step other than low-molecular-weight GTPase binding to the vesicles.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 4. Effects of nucleotides and divalent cations on recruitment of ARF to CR1 vesicles. Reaction conditions were the same as those for Figure 1 except that 10 mM CaCl2 was added in the incubation for which results are shown in lane 7. Samples were separated by SDS–12% PAGE and transferred to a nitrocellulose membrane, and ARF was detected with an alkaline phosphatase-conjugated secondary antibody. Lane 1, ARF control; lane 2, CR1 vesicles alone; lane 3, CR1 vesicles with cytosol; lane 4, CR1 vesicles with cytosol and 200 µmol/L of GDP; lane 5, CR1 vesicles with cytosol and 200 µM GTP{gamma}S; lane 6, CR1 vesicles with cytosol, 200 µM GTP{gamma}S, and 10 mM EDTA; lane 7, CR1 vesicles with cytosol, 200 µM GTP{gamma}S, and 10 mM CaCl2.

 


View larger version (18K):
[in this window]
[in a new window]
  		Figure 7. Effects of varying concentrations of phospholipid vesicles on ARF binding to immunoisolated CR1 vesicles. ARF bound to recovered CR1 vesicles was detected by enhanced chemiluminescence after separation of proteins by SDS–15% PAGE and transfer to a nitrocellulose membrane. Lane 1, ARF control; lane 2, CR1 vesicles with 400 µM GTP{gamma}S but without cytosol; lane 3, CR1 vesicles with cytosol and 400 µM GTP{gamma}S; lane 4, as in lane 3 with 56 µL of phospholipid vesicles; lane 5, as in lane 3 with 168 µL of phospholipid vesicles. The final volume of the incubation mixture was 1 mL in all cases.

 
Magnesium ion concentration and binding of ARF to CR1 storage vesicles
Although these results suggested that free Ca2+, which increases during PMN activation, did not play a role in ARF recruitment; we could not clearly discern the role of Mg2+, which is frequently involved in nucleotide binding, because it is routinely present in the "relaxation buffer" in which the CR1 vesicles are isolated. Therefore, to determine the effect of free Mg2+, we excluded Mg2+ from the "relaxation buffer" in which cells were suspended prior to the nitrogen cavitation step. Increasing amounts of ARF were recruited from the cytosol to CR1 storage vesicles when free Mg2+ was added back in increasing concentrations to the incubation buffer, as shown in Figure 5A . A plot of the densitometric scan area of ARF on the exposed film against free Mg2+ gave a value of 0.4 µM for the half-maximal effective concentration of Mg2+ in binding of ARF to CR1 vesicles (Fig. 5B) . Mg2+-EGTA buffer with varying concentrations of Mg2+ had a similar influence on ARF binding to CR1 storage vesicles, and the efficiency of ARF recruitment remained the same when conditions were adjusted to allow 10 µM free Mg2+ in the presence of either EDTA or EGTA (data not shown). The concentration of free Mg2+ is therefore a critical factor in forming a stable ARF-GTP{gamma}S-vesicle complex. The apparent enhancement of ARF binding by EDTA (Fig. 4 , lane 6) may be due to its ability to promote exchange of GTP{gamma}S for GDP bound to cytosolic ARF for the subsequent formation of a CR1 vesicle-GTP/GTP{gamma}S-Mg2+ complex. These results suggest that GTP-dependent binding of ARF to CR1 storage vesicles is a discrete process which is likely independent of the mechanisms of the increases in the intracellular free Ca2+ concentration that are necessary for up-regulation of CR1 expression on the plasma membrane during PMN activation [5 ]. None of the experiments with EDTA or EGTA caused removal of CR1 from the vesicles, suggesting that at the concentrations of these chelators used, none of the components of the reaction mixtures developed detergent-like effects.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 5. Magnesium-dependent recruitment of ARF to CR1 vesicles. (A) Western blot for ARF binding to CR1 vesicles with varying concentrations of free Mg2+ in the presence of 5 mM EDTA. Reaction conditions were the same as those described for Fig. 1 , including 400 µmol/L of GTP{gamma}S, except that Mg2+ was left out of the relaxation buffer and the buffer for each reaction was made with specific concentrations of Mg2+ in order to achieve the final free Mg2+ concentration indicated in the figure. (B) The Western blot shown in panel A was scanned by densitometer. A plot of ARF density in each lane against the calculated free Mg2+ concentration at half-maximal ARF binding gave a value of 0.4 µM for Mg2+ at 37°C.

 
Specificity of binding of ARF to CR1 storage vesicles
To determine whether the binding of ARF to CR1 vesicles is a specific interaction or whether it reflects nonspecific hydrophobic interactions, we studied the binding of ARF to traditional granules and phospholipid vesicles. We had shown earlier that a mixture of primary and secondary granules did not contain ARF and did not recruit ARF from cytosol even in the presence of GTP{gamma}S (Fig. 2B , lane 5). To provide further assurance that the binding of ARF to CR1 vesicles did not occur through nonspecific hydrophobic interactions alone, which would also allow it to interact promiscuously with other types of lipid membranes, we determined whether traditional granules would compete with CR1 vesicles for binding of ARF from the cytosol. Addition of a mixture of traditional granules, separated on 2-layer Percoll gradients, together with Mg2+ (3.5 mM) and GTP{gamma}S (400 µM), did not hinder ARF recruitment to CR1 vesicles from the cytosol (Fig. 6 , lane 4). Changing the order of addition of cytosol and granules had no observable effect (Fig. 6 , lanes 4 vs. 5 and 6 vs. 7), but inclusion of MgCl2 in the wash buffer used for tubes 6 and 7 at the end of the incubation stabilized the bound ARF so that it was more easily seen on the immunoblot. These findings further indicate that ARF does not bind to traditional granules under conditions that promote its binding to CR1 vesicles, and thus suggest that the binding to CR1 vesicles is specific.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 6. Western blot analysis of the effect of granules on ARF binding to CR1 vesicles. The conditions were same as those for Fig. 1 , including 3.5 mmol/L of MgCl2 in the incubation buffer only, except in lanes 6 and 7. Lane 1, ARF control; lane 2, CR1 vesicles alone; lane 3, CR1 vesicles with cytosol and GTP{gamma}S; lane 4, CR1 vesicles with cytosol, granules, and GTP{gamma}S; lane 5, CR1 vesicles with granules, cytosol, and GTP{gamma}S in that order of addition; lanes 6 and 7, same conditions as in lanes 4 and 5 but with 3.5 mM MgCl2 in the wash buffer as well.

 
We also used phospholipid vesicles to rule out nonspecific lipid binding of cytosolic ARF (Fig. 7 ) and to determine if ARF binding to CR1 vesicles might cause activation of PLD, as has been reported by others [10 , 14 , 22 ]. The amount of ARF bound to CR1 vesicles was not decreased by varying concentrations of phospholipid vesicles (Fig. 7 , lanes 4 and 5). The results indicate that phospholipid vesicles, like granules, do not bind ARF themselves, even in the presence of GTP{gamma}S and Mg2+, which in this experiment would be detected by inhibition of the binding of ARF to CR1 vesicles (Fig. 7 , lane 3 vs. lanes 4 and 5). We did not observe any liberation of choline, which would have indicated that ARF binding induced activation of PLD (data not shown). This led us to conclude that the binding of ARF to CR1 vesicles is not influenced by the presence of excess phospholipids, and it confirms the hypothesis that the recruitment of ARF to CR1 vesicles is not due to nonspecific hydrophobic interactions of ARF with any type of membrane lipids, but is predominantly specific and dependent on Mg2+ and GTP.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study was initiated to characterize cytosolic proteins that are capable of binding to immunoisolated CR1 storage vesicles as a first step in understanding why the movement of these vesicles differs from that of traditional granules during PMN activation. With this goal in mind, we focused on small monomeric G proteins because of their known involvement in diverse vesicular trafficking processes [6 7 8 9 10 11 12 13 14 ]. In order to study the association of these proteins with the CR1 vesicles, we took advantage of our ability to specifically immunoisolate the particular subpopulation of vesicles using antibodies against the cytoplasmic tail of CR1, as we described previously [4 ]. Although confocal microscopy might be used to detect other molecules that associate with similar vesicles, in view of the low number of CR1 molecules in a neutrophil, detection of the association of ARF with CR1 storage vesicles on stimulation would require methods such as double-label immunogold electron microscopy [25 ], which are beyond the scope of the present study. Our results show that several cytosolic proteins that are known to be involved in vesicle movement and membrane association are present on, and/or can be recruited to, isolated CR1 storage vesicles. We found that Rab3a, Rab4, and Rab5a are constitutively present on isolated CR1 storage vesicles and granules. This is consistent with others’ conclusions that most Rab proteins are localized in organelles that are involved in intracellular transport [6 ]. The functions of multiple Rabs in a single set of carrier vesicles are not well understood, but the process of recruitment of each individual Rab protein onto a single type of organelle may well be biochemically distinct, as shown for Rab7 and Rab9 binding to late endosomes [16 ]. The presence of multiple Rabs may promote shared steps of individual transport pathways and may impart specificity and directionality resulting from their interactions with each other in intracellular trafficking because of their roles in exocytic, endocytic, and transcytotic pathways [16 , 17 ]. Rabs 1a,1b, 2, and 6, for example, have also been found in transport vesicles operating in both endocytic and transcytotic pathways from rat liver fractions enriched in endoplasmic reticulum (ER)-to-Golgi transport vesicles [17 ]. CR1 storage vesicles, in this case, are presumably poised for targeting to receiving intracellular organelles and/or to the plasma membrane with a set of Rab proteins distinct from those that associate with primary and secondary granules [16 , 17 ].

We found that, unlike Rabs, one or more ARF family members can be variably recruited from the cytosol by immunoisolated CR1 storage vesicles, implying a reversible association. In many types of cells ARFs 1, 3, 4, and 5 are found in the cytosol [15 , 23 ], and neutrophils have previously been reported to contain ARFs in their cytosol [23 ]. These proteins are 84% homologous with each other, and the polyclonal antibody against human ARF1 that we used, like the monoclonal antibody ID9 [15 ], can react with multiple isoforms. Since ARF1 and ARF3 are reported to be present in greater abundance than ARF4 and ARF5 [15 ], and since our Western blots showed a single immunoreactive protein that correlated with human recombinant ARF1 protein, we believe that the major ARF protein(s) we are observing is most likely ARF1.

We investigated the conditions that favored association versus dissociation of ARF in greater detail, because we speculated that the reversibility of this interaction might be of functional significance in regulating the trafficking of these unique vesicles during cell activation in vivo. ARF is known to be required for numerous biological activities, including regulation of vesicular trafficking between intracellular organelles as well as exocytosis in other cell types [8 , 9 ].

The mechanism of exocytosis varies with cell type, and the different mechanisms leading to fusion of export vesicles with the plasma membrane may be sensitive to GTP, calcium, and/or protein phosphorylation [26 , 27 ]. Since Ca2+ is required for increased CR1 expression during PMN activation, we thought it might play a role in regulating the association of GTP-binding proteins with our isolated CR1 vesicles. In general, GTP-mediated processes require cytosol and are not inhibited by reagents that block Ca2+-mediated membrane fusion. We found that ARF protein recruitment to CR1 vesicles was enhanced by EDTA and that Ca2+ did not promote its recruitment (Fig. 4) . In contrast, with increasing concentrations of free Mg2+, there was a marked increase in the efficiency of ARF recruitment (Fig. 5) . ARF, like all other small GTP-binding proteins, is generally found free only in its GDP-Mg2+ form, whereas the GTP-Mg2+-ARF complex is the form that becomes bound to membranes. ARF goes back to its soluble form upon hydrolysis of GTP. Thus, the use of the nonhydrolyzable analog, GTP{gamma}S, stabilizes ARF in its bound form and facilitates observation of the bound complex (Fig. 2A , lane 4). A recent crystallographic study shows that the nucleotide binding site and the Mg2+ binding site are near each other and that the role of Mg2+ is to promote the nucleotide binding [27 ]. In order for ARF to be active and to bind to membranes, the bound GDP has to be replaced by GTP/GTP{gamma}S. Dissociation of GDP could be facilitated artificially either by EDTA, by different concentrations of Mg2+, or by guanine nucleotide exchange proteins in the cytosol [24 , 28 29 30 ]. This is in agreement with our findings that on regulation of the concentration of free Mg2+, more ARF was recruited to CR1 vesicles (Fig. 5) , and that the stability of the complex increased (Fig. 3 and 7) on inclusion of magnesium in the wash buffer [29 , 31 ]. The half-maximal effective concentration (0.4 µM) we observed for the effect of Mg2+ in ARF binding to CR1 vesicles is within the same order of magnitude as that reported in previous studies, showing that there is effective guanine nucleotide exchange and stable complex formation by isolated components at concentrations of free Mg2+ below 1 µM [26 , 32 ]. These results strongly suggest that GTP-dependent recruitment of ARF to CR1 storage vesicles is a distinct process independent of the fluxes in intracellular free Ca2+ concentrations that are necessary for up-regulation of CR1 expression on the plasma membrane during PMN activation [5 ].

As we found here with CR1 vesicles, ARF proteins are reported to be readily recruited to purified cellular fractions such as ER, Golgi, or endosome membranes in the presence of GTP{gamma}S, with the sole exception of ARF6 [15 ]. This is also the case with coatomer proteins, which are involved in vesicle budding [33 ]. In some studies, ARF has been shown to bind to phospholipid vesicles in the absence of other proteins [34 ]. However, our results suggest that the recruitment of ARF to CR1 storage vesicles is specific, since ARF was not contained in traditional granules and did not bind to granules or to artificial phospholipid vesicles under conditions that readily promote its association with CR1 vesicles (Fig. 6 and 7) . The composition of phospholipid vesicles we selected was designed to enable us to detect PLD if it was activated upon ARF binding [23 ], but this was not detected. If nonspecific interactions and/or lipid-lipid binding of the myristate moiety provided the predominant binding energy, we should have observed decreased ARF binding to CR1 storage vesicles in the presence of phospholipid vesicles or mixtures of traditional granules as a result of competitive binding to these other sources of lipids, but that was not the case. Since only minimal amounts of ARF were recruited in the absence of GTP{gamma}S, its binding to the CR1 vesicles is not likely due to simple electrostatic or hydrophobic interactions. Moreover, the Mg2+ dependence of the binding of ARF in the presence of GTP{gamma}S provides additional evidence that the interaction between CR1 storage vesicles and ARF is predominantly specific, although we cannot exclude the possibility that other proteins on the cytoplasmic face of CR1 vesicles, which could be absent from granule membranes, might contribute to ARF binding.

Secretory vesicles are more readily mobilized than any other compartments in neutrophils, and we have shown that once CR1 reaches the plasma membrane, it is rapidly internalized in small endocytic vesicles upon PMN activation [25 ]. Our results are consistent with the hypothesis that the interaction of CR1 storage vesicles with the plasma membrane is affected by guanine nucleotide exchange on ARFs at the site of interaction. It is possible that the physiological role of ARF binding to CR1 storage vesicles is similar to its role in the assembly of coatomer complexes on Golgi-derived vesicles for retrograde transport [35 ], and that ARF does not have a role in exocytosis. A role for ARF in endocytosis of CR1 would not be surprising, because the generation of carrier vesicles from donor membranes such as plasma membranes and/or other intracellular membranes characteristically involves GTP-dependent recruitment of coat complexes [35 , 36 ]. ARF has been associated with coat formation in other systems, for example, in assembling a multiprotein complex on the surfaces of vesicles that initiate paxillin-rich focal adhesions at the ends of prominent actin stress fibers [37 ]. Synaptic vesicle formation in neuroendocrine cell lines has been shown to occur by both clathrin-dependent and clathrin-independent pathways, and both pathways require ARF-GTP [38 ]. Previous results from our own and other laboratories suggest that CR1 can also be internalized in clathrin-dependent and clathrin-independent pathways [25 , 39 ]. Spang and Schekman [35 ] have shown that, like anterograde transport in vitro, retrograde transport from Golgi to the ER in yeast requires ARF-GTP and guanine nucleotide exchange-activating proteins (Gea1p and Gea2p). In view of our findings, it is quite conceivable that the major role of ARF in this system is to serve as a regulator of CR1 storage vesicle trafficking because of its GTP-dependent binding, and that this may be mainly involved in endocytosis rather than exocytosis. Gaynor et al. [40 ] have shown in yeast mutants that a fundamental role for ARF is to maintain membrane morphology both in ER-Golgi transport and in endocytosis. The present study establishes that ARF specifically binds to CR1 storage vesicles in a Mg2+- and GTP-dependent manner. The question of whether this is involved in anterograde or retrograde transport (i.e., exocytosis vs. endocytosis) will be the target of continuing investigation.


   ACKNOWLEDGEMENTS
 
This research was supported by National Institutes of Health grant AI22687.

Received February 21, 2001; revised May 31, 2001; accepted June 4, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sengeløv, H., Kjeldsen, L., Kroeze, W., Berger, M., Borregaard, N. (1994) Secretory vesicles are the intracellular reservoir of complement receptor 1 in human neutrophils J. Immunol. 153,804-810[Abstract]
  2. Berger, M., Wetzler, E., Welter, E., Turner, J., Tartakoff, A. (1991) Intracellular sites for storage and recycling of C3b receptors in human neutrophils Proc. Natl. Acad. Sci. USA 88,3019-3023[Abstract/Free Full Text]
  3. Borregaard, N., Heiple, J. M., Simons, E. R., Clark, R. A. (1983) Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation J. Cell Biol. 97,52-61[Abstract/Free Full Text]
  4. Kumar, A., Wetzler, E., Berger, M. (1997) Isolation and characterization of complement receptor type 1 (CR1) storage vesicles from human neutrophils using antibodies to the cytoplasmic tail of CR1 Blood 89,4555-4565[Abstract/Free Full Text]
  5. Berger, M., Medof, M. E. (1987) Increased expression of complement decay-accelerating factor during activation of human neutrophils J. Clin. Invest. 79,214-220
  6. Chavrier, P., Goud, B. (1999) The role of ARF and Rab GTPases in membrane transport Curr. Opin. Cell Biol. 11,466-475[Medline]
  7. Moss, J., Vaughan, M. (1998) Molecules in the ARF orbit J. Biol. Chem. 273,21431-21434[Free Full Text]
  8. Donaldson, J. G., Klausner, R. D. (1994) ARF: a key regulatory switch in membrane traffic and organelle structure Curr. Opin. Cell Biol. 6,527-532[Medline]
  9. Stamnes, M., Schiavo, G., Stenbeck, G., Söllner, T.H., Rothman, J. E. (1998) ADP-ribosylation factor and phosphatidic acid levels in Golgi membranes during budding of coatomer-coated vesicles Proc. Natl. Acad. Sci. USA 95,13676-13680[Abstract/Free Full Text]
  10. Houle, M., Kahn, R., Naccache, P., Bourgoin, S. (1995) ADP-ribosylation factor translocation correlates with potentiation of GTP {gamma}S-stimulated phospholipase D activity in membrane fractions of HL-60 cells J. Biol. Chem. 270,22795-22800[Abstract/Free Full Text]
  11. Schimmöller, F., Simon, I., Pfeffer, S. R. (1998) Rab GTPases, directors of vesicle docking J. Biol. Chem. 273,22161-22164[Free Full Text]
  12. Zerial, M., Stenmark, H. (1993) Rab GTPases in vesicular transport Curr. Opin. Cell Biol. 5,613-620[Medline]
  13. Nuoffer, C., Balch, W. E. (1994) GTPases: multifunctional molecular switches regulating vesicular traffic Annu. Rev. Biochem. 63,949-990[Medline]
  14. Brown, H., Gutowski, S., Moomaw, C., Slaughter, C., Sternweis, P. C. (1993) ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity Cell 75,1137-1144[Medline]
  15. Cavenagh, M., Whitney, J., Carroll, K., Zhang, C., Boman, A., Rosenwald, A., Mellman, I., Kahn, R. A. (1996) Intracellular distribution of Arf proteins in mammalian cells. Arf6 is uniquely localized to the plasma membrane J. Biol. Chem. 271,21767-21774[Abstract/Free Full Text]
  16. Soldati, T., Rancano, C., Geissler, H., Pfeffer, S. (1995) Rab7 and Rab9 are recruited onto late endosomes by biochemically distinct processes J. Biol. Chem. 270,25541-25548[Abstract/Free Full Text]
  17. Jin, M., Saucan, L., Farquhar, M. G., Palade, G. E. (1996) Rab1a and multiple other Rab proteins are associated with the transcytotic pathway in rat liver J. Biol. Chem. 271,30105-30113[Abstract/Free Full Text]
  18. Erlich, R., Gleeson, P. A., Campbell, P., Dietzsch, E., Toh, B. H. (1996) Molecular characterization of trans-Golgi p230. A human peripheral membrane protein encoded by a gene on chromosome, 6p12-22 contains extensive coiled-coil |ga-helical domains and a granin motif J. Biol. Chem. 271,8328-8337[Abstract/Free Full Text]
  19. Bascom, R. A., Srinivasan, S., Nussbaum, R. L. (1999) Identification and characterization of golgin-84, a novel Golgi integral membrane protein with a cytoplasmic coiled-coil domain J. Biol. Chem. 274,2953-2962[Abstract/Free Full Text]
  20. Subramaniam, V. N., Loh, E., Hong, W. (1997) N-Ethylmaleimide-sensitive factor (NSF) and {alpha}-soluble NSF attachment proteins (SNAP) mediate dissociation of GS28-syntaxin 5 Golgi SNAP receptors (SNARE) complex J. Biol. Chem. 272,25441-25444[Abstract/Free Full Text]
  21. Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications Proc. Natl. Acad. Sci. USA 76,4350-4354[Abstract/Free Full Text]
  22. Portezehl, H., Caldwell, P., Roegg, J. C. (1964) The dependence of contraction and relaxation of muscle fibers from the crab naia squinado on the internal concentration of free calcium Biochim. Biophys. Acta 79,581-591[Medline]
  23. Morgan, C. P., Sengeløv, H., Whatmore, J., Borregaard, N., Cockcroft, S. (1997) ADP-ribosylation-factor-regulated phospholipase D activity localizes to secretory vesicles and mobilizes to the plasma membrane following N-formylmethionyl-leucyl-phenylalanine stimulation of human neutrophils Biochem. J. 325,581-585
  24. Paris, S., Béraud-Dufour, S., Robineau, S., Bigay, J., Antonny, B., Chabre, M., Chardin, P. (1997) Role of protein-phospholipid interactions in the activation of ARF1 by the guanine nucleotide exchange factor ARNO J. Biol. Chem. 272,22221-22226[Abstract/Free Full Text]
  25. Berger, M., Wetzler, E., August, J. T., Tartakoff, A. M. (1994) Internalization of type 1 complement receptors and de novo multivesicular body formation during chemoattractant-induced endocytosis in human neutrophils J. Clin. Invest. 94,1113-1125
  26. Burgoyne, R. D., Morgan, A. (1993) Regulated exocytosis Biochem. J. 293,305-316
  27. Amor, J., Harrison, D. H., Kahn, R. A., Ringe, D. (1994) Structure of the human ADP-ribosylation factor 1 complexed with GDP Nature 372,704-708[Medline]
  28. Franco, M., Chardin, P., Chabre, M., Paris, S. (1993) Myristoylation is not required for GTP-dependent binding of ADP-ribosylation factor ARF1 to phospholipids J. Biol. Chem. 268,24531-24534[Abstract/Free Full Text]
  29. Béraud-Dufour, S., Robineau, S., Chardin, P., Paris, S., Chabre, M., Cherfils, J., Antonny, B. (1998) A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the ß-phosphate to destabilize GDP on ARF1 EMBO J 17,3651-3659[Medline]
  30. Antonny, B., Béraud-Dufour, S., Chardin, P., Chabre, M. (1997) N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange Biochemistry 36,4675-4684[Medline]
  31. Franco, M., Chardin, P., Chabre, M., Paris, S. (1996) Myristoylation-facilitated binding of the G-protein ARF1GDP to membrane phospholipids is required for its activation by a soluble nucleotide exchange factor J. Biol. Chem. 271,1573-1578[Abstract/Free Full Text]
  32. Knight, D. E., von Grafenstein, H., Athayde, C. M. (1989) Calcium-dependent and calcium-independent exocytosis Trends Neurosci 12,451-458[Medline]
  33. Whitney, J. A., Gomez, M., Kreis, T. E., Mellman, I. (1995) Cytoplasmic coat proteins involved in endosome function Cell 83,703-713[Medline]
  34. Randazzo, P. A., Terui, T., Sturch, S., Fales, H. M., Ferrige, A. G., Khan, R. A. (1995) The myristoylated amino terminus of ADP-ribosylation factor 1 is a phospholipid- and GTP-sensitive switch J. Biol. Chem. 270,14809-14815[Abstract/Free Full Text]
  35. Spang, A., Schekman, R. (1998) Reconstitution of retrograde transport from the Golgi to the ER in vitro J. Cell Biol. 143,589-599[Abstract/Free Full Text]
  36. Schekman, R., Orci, L. (1996) Coat proteins and vesicle budding Science 271,1526-1533[Abstract]
  37. Norman, J. C., Jones, D., Barry, S. T., Holt, M. R., Cockcroft, S., Critchley, D. R. (1998) ARF mediates paxillin recruitment to focal adhesions and potentiates Rho-stimulated stress fiber formation in intact and permeabilized Swiss 3T3 fibroblasts J. Cell Biol. 143,1981-1995[Abstract/Free Full Text]
  38. Shi, G., Faundez, V., Roos, J., Dell’Angelica, C., Kelly, R. (1998) Neuroendocrine synaptic vesicles are formed in vitro by both clathrin-dependent and clathrin-independent pathways J. Cell Biol. 143,947-955[Abstract/Free Full Text]
  39. Abrahamson, D. R., Fearon, D. T. (1983) Endocytosis of C3b receptor of complement within coated pits in human polymorphonuclear leukocytes and monocytes Lab. Invest. 48,162-168[Medline]
  40. Gaynor, E. C., Chen, C.-Y., Emr, S. D., Graham, T. R. (1998) ARF is required for maintenance of yeast Golgi and endosome structure and function Mol. Biol. Cell 9,653-670[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. J. Herrero-Turrion, J. Calafat, H. Janssen, M. Fukuda, and F. Mollinedo
Rab27a Regulates Exocytosis of Tertiary and Specific Granules in Human Neutrophils
J. Immunol., September 15, 2008; 181(6): 3793 - 3803.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chaudhuri, S.
Right arrow Articles by Berger, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chaudhuri, S.
Right arrow Articles by Berger, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS