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


     

Originally published online as doi:10.1189/jlb.0203066 on July 22, 2003

Published online before print July 22, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0203066v1
74/5/800    most recent
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 Google Scholar
Google Scholar
Right arrow Articles by Rosén, H.
Right arrow Articles by Olsson, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rosén, H.
Right arrow Articles by Olsson, I.
(Journal of Leukocyte Biology. 2003;74:800-809.)
© 2003 by Society for Leukocyte Biology

Artificially controlled aggregation of proteins and targeting in hematopoietic cells

Hanna Rosén1, Ying Gao, Ellinor Johnsson and Inge Olsson

Institution for Laboratory Medecine, Department of Hematology, Lund, Sweden

1Correspondence: Institution for Laboratory Medecine, Department of Hematology, C14, BMC, S-221 84, Lund, Sweden. E-mail: Hanna.Rosen{at}hematologi.lu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The targeting mechanisms for granule proteins in hematopoietic cells are largely unknown. Aggregation is believed to be important for protein sorting-for-entry and sorting-by-retention in endocrine and neuroendocrine cells. We asked whether artificially induced multimerization/aggregation of chimeric proteins could affect their sorting in hematopoietic cells. A system was used that permits ligand-controlled intracellular oligomerization of hybrid proteins containing the FK506-binding protein (FKBP). The hybrid proteins ELA-(FKBP)3 with neutrophil elastase (ELA) and (FKBP*)4-FCS-hGH with a furin cleavage site (FCS) and human growth hormone (hGH) were expressed in the myeloblastic 32D and the rat basophilic leukemia (RBL-1) hematopoietic cell lines. ELA alone is normally targeted to secretory lysosomes. However, the hybrid proteins and ligand-induced aggregates of them were constitutively secreted and not targeted. The hGH that was released at the FCS in (FKBP*)4-FCS-hGH was also constitutively secreted. We conclude that protein multimerization/aggregation per se is not enough to facilitate sorting-for-entry to secretory lysosomes in hematopoietic cells and that improperly folded proteins may be eliminated from sorting by constitutive secretion.

Key Words: secretory lysosomes • quality control • multimerization • secretion • storage


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The secretory pathway accomplishes protein sorting by moving cargo between membrane-bound compartments of cells with the aid of vesicular carriers [1 ]. In cells, this path has a distal route for constitutive secretion. In addition, specialized cells such as endocrine, exocrine, and hematopoietic cells have an itinerary for retrieval and targeting for storage organelles [2 3 4 ]. Vesicular carriers form on the trans-Golgi network (TGN) donor membranes and package cargo together with proteins that are required in the targeting system. Precursor cells of hematopoietic origin synthesize subsets of storage granules with a unique composition [5 , 6 ]. These granules can be stimulated to release their bioactive agents into the phagocytic vacuole or exterior milieu to participate in host defense. The mannose-6-phosphate receptor (MPR) system [7 ] targets lysosomal hydrolases and granzymes in cells such as natural killer cells and cytotoxic T-lymphocytes [8 ]. However, this receptor does not seem to be necessary for selection of proteins destined for granules in neutrophil precursors [6 ]. A specific sorting mechanism for each granule subtype may not be required, as the genes that are expressed during a particular window of differentiation limit the composition of granules [9 ]. However, retrieval is required for the rescue of newly synthesized protein from constitutive secretion [6 ]. Here, we attempted to investigate the role of protein multimerization/aggregation for retrieval and targeting in hematopoietic cells.

Endocrine and exocrine cells have granules for regulated secretion that are released extracellularly upon stimulation. In these cells, proteins are routed to secretory vesicles within the TGN (sorting-for-entry) that form immature granules from which mis-sorted lysosomal enzymes are removed and re-routed to the lysosomal pathway [2 , 4 , 10 , 11 ]. Protein processing and condensation are completed in the mature granules (sorting-by-retention) [2 , 4 ]. A low pH and a millimolar concentration of calcium have been suggested to facilitate aggregation in the TGN [4 , 12 ]. By contrast, hematopoietic cells have secretory lysosomes that combine storage, regulated secretion, and lysosomal functions. Their formation may involve an endosomal and a direct route from TGN [13 , 14 ]. Azurophil granules of neutrophils are also lysosome-like, although they lack certain lysosome-associated membrane proteins (LAMPs) such as LAMP-1 and LAMP-2 [15 , 16 ] but do contain CD63/LAMP-3 [13 , 17 ]. Secretory lysosomes of hematopoietic cells appear to be defective in the Chediak-Higashi syndrome [13 , 14 ]. This deficiency indicates that secretory lysosomes belong to a common lineage that is affected specifically in this syndrome. Secretory lysosomes can have electron-dense cores and multivesicular regions [14 , 18 , 19 ]. Proteins might be sorted into these two regions by separate pathways. In rat basophilic leukemia (RBL) cells, evidence indicates that proteins may be targeted through a MPR-dependent endosomal pathway or a MPR-independent, nonendosomal pathway or both [20 ].

The aggregation properties of granule proteins have been suggested to be important in sorting-for-entry upon Golgi exit or sorting-by-retention in the mature granules. We therefore investigated a role for aggregation in protein targeting to secretory lysosomes of the hematopoietic 32D c13 [21 ] and RBL-1 [22 ] cell lines. We asked whether artificially induced aggregation of proteins affected their sorting within the hematopoietic cells. A system was used that permitted analysis of intracellular protein oligomerization that could be reversibly controlled by derivatives of the lipid-soluble and cell-permeable drug FK506 [23 ]. Two fusion proteins were expressed: ELA-(FKBP)3, which consisted of neutrophil elastase (ELA) and three copies of the human FKBP, and (FKBP*)4-FCS-hGH, which consisted of four copies of mutated FKBP (FKBP*), a furin cleavage site (FCS) and human growth hormone (hGH) [24 ]. In contrast to FKBP-containing aggregates, FKBP*-containing aggregates are formed spontaneously and are dissolved by a monomeric ligand. The aim was to determine whether ligand-controlled aggregation would affect targeting for secretory lysosomes. Our results indicate that protein multimerization/aggregation is not enough to facilitate sorting-for-entry to secretory lysosomes in hematopoietic cells. We speculate that targeting may require a native conformation, and non-native proteins may be eliminated from sorting by constitutive secretion as part of quality control.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
The eukaryotic expression vector pcDNA3.1(–) was from Invitrogen (Groningen, The Netherlands). ARIAD Pharmaceuticals (University Park, Cambridge, MA) supplied the expression plasmids pC4M-FV2E, pC4M-FV1E, and pC4S1-FM4-FCS-hGH as well as the FKBP ligands AP20187 (dimeric ligand) and AP21998 (monomeric ligand). [35S]Methionine/[35S]cysteine (cell radiolabeling grade) was from ICN Biomedicals (Irvine, CA). Percoll was from ICN Biomedicals (Aurora, OH). Heat-inactivated fetal bovine serum (FBS), Iscove’s modified Dulbecco’s medium (IMDM) with glutamax, and methionine cysteine-free RPMI-1640 medium were from Gibco-BRL (Life Technologies, Rockville, MD). IMDM (1x) with L-glutamine and RPMI 1640 was from PAA Laboratories (GmbH, Austria). The TNT® T7 coupled reticulocyte lysate system was from Promega (Madison, WI). Protein A-Sepharose CL-4B was from Amersham Pharmacia Biotech (Uppsala, Sweden). Protein G-Sepharose was from Santa Cruz Biotechnology (Santa Cruz, CA). Geneticin and CompleteTM protease inhibitor cocktail tablets were from Roche Diagnostics GmbH (Mannheim, Germany). Endoglycosidase H- and N-glycosidase were from Roche Diagnostics Scandinavia AB (Bromma, Sweden). ECLTM and HybondTM ECLTM nitrocellulose membranes were from Amersham Pharmacia Biotech, UK Ltd. (Buckinghamshire). Precast gels (10–20%) were from Novex (San Diego, CA). The Dounce glass homogenizer was from Kontes (Vineland, NJ). Rabbit polyclonal antibodies against FKBP12 were from BIOMOL Research Laboratories (Plymouth Meeting, PA), mouse monoclonal antibodies against human neutrophil ELA were from BD PharMingen (San Diego, CA), and rabbit polyclonal antibodies against hGH were from DAKO (Carpinteria, CA). The secondary antibodies used in the Western blots, goat anti-rabbit immunoglobulin G (IgG), and goat anti-mouse IgG (H+L)-horseradish peroxidase conjugate were from Bio-Rad Laboratories (Hercules, CA).

The following buffer systems were used: homogenization buffer (0.25 M sucrose, 0.5 mM NaEDTA, 10 mM HEPES, pH 7.3), cell lysis buffer [1 M NaCl, 50 mM Tris-HCl, pH 8.0, 0.5% Triton X-100 (v/v)], radioimmune precipitation buffer [RIPA; 0.75 M NaCl, 0.15 M HEPES, pH 7.3, 0.5% sodium dodecyl sulfate (SDS; v/v), 5% Triton X-100 (v/v), 5% sodium deoxycholate (w/v)]. Protease inhibitors were added to the above buffers before use. Immunoblotting lysis buffer contained 86 mM Tris (pH 6.8), 11% (v/v) glycerol, 2.3% (w/v) SDS, 1.2% (v/v) ß-mercaptoethanol, and 0.005% (v/v) bromophenol blue, and electrophoresis sample buffer contained 0.4 M Tris-HCl, pH 6.8, 50% (v/v) glycerol, 10% (w/v) SDS, and 5% (v/v) ß-mercaptoethanol.

Construction of expression vectors
The ELA-(FKBP)3 was constructed in pcDNA3 or pcDNA3.1/-. A FKBP fragment was excised from pC4FV1E by digestion with Xba1 and BamHI and ligated into pC4M-FV2E from which the hemagglutinin epitope had been eliminated by digestion with Spe1 and BamHI. (FKBP)3 was excised by digestion with Xba1 and BamHI and ligated into pcDNA3.1/– to create pcDNA3.1/-(FKBP)3. ELA was amplified by polymerase chain reaction with neutrophil ELA cDNA [25 ] as a template. The following primers were used to create flanking restriction enzyme sites for Apa1 and Xba1: upstream 5'-TTCGGAGGGCCCGCCACCATGACCCTCGGCCGCC-GACTCGC and downstream 5'-TTCGGATCTAGAGTGGGTCCTGCTGGCC-GGGTC. The product was ligated into pcDNA3.1/-(FKBP)3 to create pCDNA3.1/-ELA-(FKBP)3, abbreviated ELA-(FKBP)3. The in vitro-translated protein showed the expected molecular size.

The expression plasmid pC4S1-FM4-FCS-hGH contains the sequences for 4 FM (FKBP/F36M/mutant), FCS, and hGH. It also contained a signal sequence from hGH. The cDNA insert encoding the fusion protein FM4-FCS-hGH was cut out with EcoRI and BamHI and ligated into pcDNA3.1(–) to create the expression vector pcDNA3-FM4-FCS-hGH, designated (FKBP*)4-FCS-hGH. The in vitro-translated protein showed the expected molecular size.

Cell culture
The rat basophilic/mast cell line RBL-1 [22 ] was grown in RPMI 1640 supplemented with 10% heat-inactivated FBS. The murine myeloblast-like 32D c13 cell line [21 ] was grown in IMDM with L-glutamin, 10% heat-inactivated FBS, and 30% WEHI-conditioned medium as a source of interleukin-3 [26 ]. The cell cultures were kept in 5% CO2 at 37°C in a fully humidified atmosphere. Exponentially growing cells were used in all the experiments.

Transfection
The cells were transfected by addition of 15 µg plasmid to 4 x 106 cells in 400 µl complete medium, using the Bio-Rad electroporation apparatus, with electrical settings of 960 µF and 260 V. After electroporation, 5 x 105 cells/ml were incubated for 48 h to allow expression of the geneticin resistance. Geneticin was then added at a concentration of 1 mg/ml to a maximum of 5000 cells/well in 100 µl complete medium in 96-well plates. Individual antibiotic-resistant cell clones were expanded in suspension cultures and screened for the expression of the transfected cDNA.

Biosynthetic radiolabeling
Cells (2x106/ml) were starved for 30 min in methionine, cysteine-free RPMI-1640 medium, supplemented with 10% dialyzed FBS, to deplete the intracellular pool of methionine and cysteine and thus facilitate subsequent incorporation of radioactive amino acids. The cells were then incubated for 30 or 60 min in identical medium supplemented with [35S]methionine/[35S]cysteine (25 µCi/ml) to allow radiolabeling of de novo-synthesized proteins (pulse radiolabeling). For radiolabel chase, to follow changes as proteins progressed through the secretory pathway, the cells were resuspended in complete medium at a density of 2 x 106/ml. At timed intervals, cells were withdrawn and subjected to extraction by cell lysis buffer or homogenization and subsequent subcellular fractionation.

Subcellular fractionation
Subcellular fractionation was performed on continuous gradients of Percoll, as described previously [27 , 28 ]. All steps were performed at 4°C. Briefly, 1 x 108 cells were resuspended in homogenization buffer containing protease inhibitors and homogenized with 15–25 strokes using a Dounce glass homogenizer. The postnuclear supernatant obtained by centrifugation at 500 gfor 10 min was centrifuged in a 20% Percoll density gradient. Nine fractions were collected, and all the cytosol appeared in fraction 9 [27 , 28 ]. The peak activities of ß-hexosaminidase, fractions 1–2, and galactosyl transferase, fraction 6, indicate the positions in RBL-1 cells of lysosomes and Golgi elements, respectively [25 ]. The peak activities of ß-hexosaminidase and galactosyl transferase in Percoll fractions of 32D c13 cells were localized in fractions 2 and 6, respectively [29 ].

Immunoprecipitation (IP)
All steps were performed at 4°C. Cells (2x106/ml) were solubilized in cell lysis buffer. Percoll-containing subcellular fractions were lysed with 1 vol H2O and 1/2 vol fivefold-concentrated RIPA. The cell lysates were frozen and thawed and cleared by centrifugation at 32,000 g for 60 min at 4°C. IP was performed in a mixture of antiserum, 5 mg/ml protein A-Sepharose, and 0.2% (v/v) protein G-agarose solution, and the mixture was rotated overnight. The pellet was recovered and washed three times with RIPA, dissolved in electrophoresis sample buffer, and boiled for 5 min, whereupon SDS-polyacrylamide gel electrophoresis (PAGE) was performed in a precast, 10–20% Tris-glycine gel. The gels were exposed to X-ray film at –80°C, or immunoblotted as described below. Densitometry measurements were performed in a Molecular Imager® FX (Bio-Rad Laboratories), and linear data were obtained by avoiding overexposure.

Immunoblotting
The enhanced chemiluminescence (ECL) Western blotting kit was used according to the manufacturer’s instructions. For direct immunoblotting, cells (5x106) were washed in phosphate-buffered saline and sonicated in 100 µl lysis buffer for immunoblotting, boiled for 5 min, and cleared by centrifugation at 14,000 g at 4°C for 5 min. A lysate from 0.5 x 106 cells was loaded in each lane of a precast, 10–20% Tris-glycine gel. Recovered pellets of immunoprecipitated protein from subcellular fractionation were dissolved in 100 µl immunoblotting lysis buffer and boiled for 5 min. Protein samples corresponding to 2 x 106 cells were loaded in each lane of a precast, 10–20% Tris-glycine gel. Proteins were electrophoretically transferred to Hybond-P nitrocellulose membrane in blotting buffer. Detection was performed by exposing the membranes to HyperfilmTM ECLTM for 10–30 s.

Gel filtration chromatography
Molecular size was determined by gel filtration chromatography on a Superose 6 column. Cell extracts in cell lysis buffer were run on the column equilibrated in the same buffer followed by IP of the fractions with anti-ELA.

Digestion with glycosidases
An immunoprecipitate corresponding to 20 x 106 cells was incubated with 0.24 U endoglycosidase H (endo-H) or 2 U N-glycosidase at 37°C for 24 h [27 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experimental outline
The rationale for the experiments was to determine whether protein aggregation may affect targeting in the secretory pathway of hematopoietic cells. The approach is based on a system where reversible aggregation of the protein FKBP12, normally a cytosolic protein [30 ], is controlled pharmacologically with cell-permeable ligands [23 , 31 ]. Dimeric ligand (AP20187) induces aggregation of FKBP in ELA-(FKBP)3, and monomeric ligand (AP21998) dissolves preformed aggregates of FKBP* in (FKBP*)4-FCS-hGH. (FKBP*)4-FCS-hGH has been used for pharmacological regulation of protein secretion through monomeric, ligand-controlled aggregation in the endoplasmic reticulum (ER) [24 ].

The hybrid protein ELA-(FKBP)3 contains neutrophil ELA with its N-terminal translocation signal peptide adjacent to three copies of FKBP. ELA has been shown to be targeted to granules when expressed in the cell lines used [25 ]. The addition of dimeric ligand is assumed to initiate aggregation among ELA-(FKBP)3 molecules. (FKBP*)4-FCS-hGH consists of an N-terminal translocation signal peptide (from growth hormone) followed by four copies of FKBP* to allow for the spontaneous formation of large aggregates, a FCS, and finally, hGH [24 ]. The FCS will allow furin-catalyzed release of hGH in the Golgi where furin is located [32 ].

Stable expression of the constructs in the hematopoietic cell lines should lead to the synthesis and translocation of the hybrid proteins into the lumen of the ER followed by export to the Golgi of a protein in its native conformation. The protein may then become constitutively secreted or targeted to secretory lysosomes. With use of the fusion proteins, it should be possible to investigate whether the aggregation state per se will affect the targeting to secretory lysosomes.

Efficiency of extraction and IP of hybrid proteins
To compare extraction efficiencies, 32D cells expressing ELA-(FKBP)3 were incubated with or without 10 nM dimeric ligand for 2 days followed by extraction with RIPA or cell lysis buffer. The extracts were analyzed by IP-Western, and direct Western analyzed the pelleted remnants. The extraction efficiency was similar with RIPA and cell lysis buffer (data not shown). In addition, there was no indication of aggregate precipitation without antibody that should have been reflected in a larger loss in the pelleted remnants in the presence of dimeric ligand. To determine the effect of protein aggregation on the IP efficiency, extracts were prepared from 32D cells after 1 h radiolabeling and 2 h radiolabel chase. RIPA extracts and lysis buffer extracts were incubated separately overnight at room temperature with 10 nM dimeric ligand (to induce aggregation) or without ligand and were subsequently immunoprecipitated with anti-ELA. The amount of immunoprecipitate as detected by SDS-PAGE did not differ between samples with or without dimeric ligand (data not shown). Consequently, monomeric and aggregated ELA-(FKBP)3 are immunoprecipitated with equal efficiency.

Controlled aggregation of ELA-(FKBP)3
32D and RBL cells that expressed ELA-(FKBP)3 were incubated with various concentrations of dimeric ligand to assess the effect on retention and secretion of the protein. The results from IP-Western and radiolabeling indicated that a critical dimeric ligand concentration of 10 nM or higher promoted intracellular accumulation of ELA-(FKBP)3 (Fig. 1A ). A pulse-chase experiment with various chase times was also performed (Fig. 1B) . The results showed a marked ligand-induced accumulation of ELA-(FKBP)3. However, densitometry data show the relative cellular retention of newly synthesized ELA-(FKBP)3 to be similar for cells with or without added dimeric ligand (Fig. 1C) . Relative secretion is somewhat higher for cells with dimeric ligand than for cells without. In RBL cells, dimeric ligand concentration of 10 nM did not affect the accumulation of the endogenous granule protein, rat mast cell protease II, indicating that this ligand did not interfere with normal processing (data not shown).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 1. Ligand titration. Dimeric ligand titration was performed in RBL (A, I and II) and 32D cells (A, III) expressing ELA-(FKBP)3. (A, I) RBL cells were incubated for 48 h in the presence of various concentrations of dimeric ligand followed by immunoblotting of cell extracts with anti-FKBP. (A, II and III) RBL (A, II) and 32D (A, III) cells were radiolabeled (pulse) in the presence of various concentrations of dimeric ligand for 3 h followed by extraction and IP with anti-ELA of cell extracts and culture medium. (B) 32D cells expressing ELA-(FKBP)3 were radiolabeled for 30 min with or without 10 nM dimeric ligand, followed by radiolabel chase under the same conditions. At depicted time points, cells were removed, extracted, and immunoprecipitated with anti-ELA. The position of the ELA-(FKBP)3 is indicated to the right with arrows. (C) Densitometric results show the relative accumulation and secretion of ELA-(FKBP)3 after chase start (0), at which time the value is set to 100%. The secretion of ELA-(FKBP)3 during the 30-min radiolabeling was negligible (data not shown) and therefore, indicated as 0% in this diagram.

 
Results from gel filtration chromatography of 32D cell extracts obtained after 1 h radiolabeling followed by a 2-h radiolabel chase demonstrated that 10 nM dimeric ligand induced multimer formation (Fig. 2 ). The molecular size of monomeric ELA-(FKBP)3 corresponded to ~60 kDa, and the ligand-induced multimers showed a very broad elution range with multimers of 600 kDa and higher.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. Dimeric ligand-induced aggregate formation as shown by gel filtration. 32D cells expressing ELA-(FKBP)3 were radiolabeled for 60 min (pulse) followed by a 2-h radiolabel chase without (–) or with (+) 10 nM dimeric ligand. Aliquots of cell extracts in cell lysis buffer were run on a Superose 6 column equilibrated in the same buffer, followed by IP of the eluted fractions with anti-ELA. Arrows indicate elution volumes for molecular weight (Mr) 669,000 thyroglobulin, Mr 440,000 ferritin, and Mr 67,000 bovine serum albumin. The position of the ELA-(FKBP)3 is indicated to the right with arrows. The presence of dimeric ligand (+) resulted in formation of multimers with a much higher molecular size as compared with the monomer seen in the absence of the ligand (–).

 
ELA-(FKBP)3 glycoforms
Newly synthesized ELA-(FKBP)3 was sensitive to digestion with endo-H, indicating the presence of high mannose glycoforms that are produced in the ER (Fig. 3 ). However, 60 min pulse radiolabeling also resulted in the production of some endo-H-resistant ELA-(FKBP)3 through generation of complex oligosaccharide side-chains (Fig. 3) . The fraction of endo-H-resistant oligosaccharide side-chains increased with radiolabel chase, indicating Golgi-derived, complex oligosaccharide side-chain formation. This was supported by densitometric readings that showed a ratio between endo-H-resistant (complex oligosaccharide side-chains) and susceptible (high mannose oligosaccharide side-chains) ELA-(FKBP)3 of 1.2, 3.5, and 3.5 for pulse, chase plus dimeric ligand, and chase minus dimeric ligand samples, respectively. These results confirm that most of the newly synthesized, high mannose ELA-(FKBP)3 was exported from the ER and converted to ELA-(FKBP)3 with complex oligosaccharides in the Golgi. The secreted ELA-(FKBP)3 was endo-H-resistant, indicating side-chain modification during passage through Golgi. Addition of dimeric ligand did not affect the endo-H sensitivity.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 3. Oligosaccharide side-chains. 32D cells expressing ELA-(FKBP)3 were radiolabeled for 60 min (pulse) with dimeric ligand (+) followed by radiolabel chase for 60 min without (–) or with (+) the dimeric ligand. Cell lysates and medium were immunoprecipitated with anti-ELA followed by digestion with endo-H or N-glycanase. ELA-(FKBP)3 formed a double band. Pulse and chase samples showed an endo-H-susceptible fraction of ELA-(FKBP)3 with high mannose oligosaccharide side-chains of the same size as the N-glycanase digestion product. Densitometric readings (not given) showed a ratio between endo-H-resistant and -susceptible ELA-(FKBP)3 of 1.2, 3.5, and 3.5 for pulse, chase + dimeric ligand, and chase – dimeric ligand samples, respectively. The fraction of endo-H-resistant oligosaccharide side-chains increased with radiolabel chase, indicating Golgi-derived, complex, oligosaccharide side-chain formation. The ELA-(FKBP)3 of the incubation medium was endo-H-resistant. The results depicted are from a Molecular Imager® FX (Bio-Rad).

 
Subcellular distribution of ELA-(FKBP)3
To investigate whether the aggregation status of ELA-(FKBP)3 influenced its intracellular location under "steady-state" conditions, subcellular fractionation followed by IP and immunoblotting was performed in 32D cells (Fig. 4 ). Cells expressing the hybrid protein were incubated without (Fig. 4A) or with (Fig. 4B) 10 nM dimeric ligand for 48 h. In both cases, the fusion protein showed a broad distribution without significant accumulation in the densest, subcellular fractions. The densitometric readings showed the relative distribution in the densest fractions 1–3 to be 10% higher in the presence of dimeric ligand than in its absence. In conclusion, this minimal accumulation of ELA-(FKBP)3 in dense fractions does not support significant targeting to secretory lysosomes by ligand-induced aggregation.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 4. Intracellular steady-state distribution of ELA-(FKBP)3. 32D cells (100x106) expressing ELA-(FKBP)3 were incubated for 48 h without (A) or with (B) 10 nM dimeric ligand. The cells were homogenized, and the postnuclear supernatant was subcellularly fractionated by centrifugation on a Percoll gradient. Extracts of the fractions were immunoprecipitated with anti-ELA and analyzed by immunoblotting with anti-FKBP as described in Materials and Methods. The position of the ELA-(FKBP)3 is indicated to the right with arrows. Densitometric readings of the ELA-(FKBP)3 band are shown as % of total. The peak activities of the secretory lysosome marker ß-hexosaminidase and the Golgi marker galactosyl transferase were in fraction 2 and fraction 6, respectively [29 ]. Dashed arrows indicate the position of these markers.

 
Processing of the (FKBP*)4-FCS-hGH
The processing was investigated in RBL cells (Fig. 5 ) and 32D cells (Fig. 6 ) by pulse-chase radiolabeling. Three conditions were tested: no ligand present (– –), monomeric ligand present during pulse but not radiolabel chase (+ –), and monomeric ligand during pulse and chase (+ +). Under the – – condition, the hybrid protein self-associated in the ER [24 ]. Under the + – condition, the aggregates dissolved but may have the ability to reform during the chase when monomeric ligand is removed. Under the + + condition, aggregates should remain dissolved during the entire experiment. The proteins were immunoprecipitated with anti-hGH, which detects (FKBP*)4-FCS-hGH and hGH generated by cleavage at the FCS site.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 5. Processing of (FKBP*)4-FCS-hGH in RBL cells. Cells expressing (FKBP*)4-FCS-hGH were incubated with [35S]methionine/[35S]cysteine for 30 min (pulse) without (A) or with (B, C) 1 µM monomeric ligand, followed by radiolabel chase without (A, B) or with (C) the same ligand for up to 4 h. At depicted time points, 20 x 106 cells were removed and after lysis, immunoprecipitated with anti-hGH and were analyzed as described in Materials and Methods. The positions of the (FKBP*)4-FCS-hGH and cleaved-off hGH are indicated to the right with arrows. Numbers to the left are the values of molecular mass standards.

 


View larger version (33K):
[in this window]
[in a new window]
  		Figure 6. Processing of (FKBP*)4-FCS-hGH in 32D cells. Cells expressing (FKBP*)4-FCS-hGH were incubated with [35S]methionine/[35S]cysteine for 30 min (pulse) without (A) or with (B, C) 1 µM monomeric ligand, followed by chase of the radiolabel without (A, B) or with (C) the same ligand for up to 4 h. At depicted time points, 20 x 106 cells were removed and after lysis, immunoprecipitated with anti-hGH and were analyzed as described in Materials and Methods. The positions of the (FKBP*)4-FCS-hGH and cleaved-off hGH are indicated to the right with arrows. Numbers to the left are the values of molecular mass standards.

 
No processing occurred in the absence of monomeric ligand in RBL cells (– –), secretion was negligible, and the hybrid protein was retained and degraded (Fig. 5A) . Condition (+ –) gave rise to some hybrid protein processing, resulting in hGH formation as well as promoting an increase in the secretion of hybrid protein and hGH; cell retention of cleaved hGH was minimal (Fig. 5B) . Condition (+ +) gave similar results as + – (Fig. 5C) . On the basis of densitometric analyses, the ratio between secreted and intracellular (FKBP*)4-FCS-hGH at 1-h chase was 0.02 in – –, 0.4 in + –, and 0.3 in + +. The corresponding values for the ratio between secreted hGH and intracellular (FKBP*)4-FCS-hGH at 1-h chase were 0, 0.1, and 0.03.

In contrast to RBL cells, 32D cells showed some hybrid protein and hGH secretion even without addition of monomeric ligand (Fig. 6A) . Condition + – increased the hybrid protein processing as well as hybrid protein and hGH secretion (Fig. 6B) . Condition + + resulted in more pronounced processing and hGH secretion compared with + – (Fig. 6C) . On the basis of densitometric analyses, the ratio between secreted and intracellular (FKBP*)4-FCS-hGH at 1-h chase was 0.1 (– –), 0.6 (+ –), and 0.4 (+ +). The corresponding values for the ratio between secreted hGH and intracellular (FKBP*)4-FCS-hGH at 1-h chase were 0.02, 0.3, and 1.4. These analyses show that the most pronounced secretion takes place when monomeric ligand is present during pulse and chase.

Subcellular distribution of (FKBP*)4-FCS-hGH
To investigate whether the aggregation status of (FKBP*)4-FCS-hGH influenced its intracellular location under steady-state conditions, similar experiments to those described above for ELA-(FKBP)3 (Fig. 4) were performed with (FKBP*)4-FCS-hGH (Fig. 7 ). 32D cells expressing this hybrid protein were incubated without (Fig. 7A) or with (Fig. 7B) 1 µM monomeric ligand AP21998 for 48 h. The cells were then homogenized and subcellularly fractionated followed by IP and immunoblotting. (FKBP*)4-FCS-hGH showed a broad distribution without (Fig. 7A) and with ligand (Fig. 7B) , without significant accumulation in the most-dense, subcellular fractions. Cleaved-off hGH could be observed in light-density fractions (Fig. 7A and 7B) . These results suggest a lack of significant targeting of the (FKBP*)4-FCS-hGH hybrid protein or released hGH to the secretory lysosomes in these cells. In addition, these experiments have also been performed in RBL cells with similar results, which were confirmed by immunoelectron microscopy (data not shown).



View larger version (60K):
[in this window]
[in a new window]
  		Figure 7. Intracellular steady-state distribution of (FKBP*)4-FCS-hGH. 32D cells (100x106) expressing (FKBP*)4-FCS-hGH were incubated for 48 h without (A) or with (B) 1 µM monomeric ligand. The cells were homogenized, and the postnuclear supernatant was subcellularly fractionated by centrifugation on a Percoll gradient. Extracts of the fractions were immunoprecipitated with anti-hGH and analyzed by immunoblotting with anti-hGH. The positions of (FKBP*)4-FCS-hGH and cleaved-off hGH are indicated to the right with arrows. Numbers to the left are the values of molecular mass standards. The peak activities of the secretory lysosome marker ß-hexosaminidase and the Golgi marker galactosyl transferase were in fraction 2 and fraction 6, respectively [29 ]. Dashed arrows indicate the position of these markers.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Active and passive sorting
An active sorting postulates that proteins destined for granule targeting bind to sorting receptors that have molecular addresses for selective delivery of cargo to granules. To accomplish this, cargo proteins need a signal for binding to the receptors. However, with the exception of mannose-6-phosphate-specific sorting, signals have not been identified in proteins destined for the granule matrix. After binding to a specific receptor, this signal directs the targeting of hydrolytic enzymes to lysosomes in many cells and granzymes to secretory lysosomes in hematopoietic cells [33 ]. Conversely, transmembrane proteins such as LAMP 1 and 2 and CD 63 (LAMP 3), which are destined for the secretory lysosome membrane, have highly specific sorting signals within their cytoplasmic amino acid sequences [34 ]. A lack of these molecular addresses would lead to constitutive secretion by default. In contrast, a passive sorting process postulates that delivery to granules is without selectivity and independent of receptor binding. Granule proteins, whose synthesis is synchronized, become colocalized in granules as a result of the timing of gene expression, which determines the composition of granule subtypes [5 ]. In addition, a mechanism is needed for retrieval to avoid constitutive secretion.

The aggregation state of a protein is believed to be important for targeting in secretory granules of endocrine and neuroendocrine cells [10 , 11 ]. In these cells, protein aggregation has been suggested to stimulate the formation of immature secretory granules in the TGN (sorting-for-entry), and subsequent remodeling into mature granules occurs after the retrieval of lysosomal proteins from the immature granules (sorting-by-retention). Regulated secretory proteins to be sorted may interact, in an aggregated form, with membrane microdomains rich in sphingolipids [4 ]. The present experiments were performed to test the importance of induced aggregation for targeting in hematopoietic cells. Results from gel filtration showed that high molecular weight multimers/aggregates were formed upon ligand incubation of cells with ELA-(FKBP)3 (Fig. 2) . Moreover, results have shown that (FKBP*)4-FCS-hGH is capable of forming large aggregates in cells [24 ].

Our results did not support a direct role for aggregation in sorting-for-entry in the hematopoietic cell lines. Thus, aggregation of the hybrid proteins did not promote targeting to secretory lysosomes. However, aggregation is assumed to be important in sorting by forming the typical dense cores of the secretory lysosomes, corresponding to sorting-by-retention. It was not possible to shed any light on this latter process as a result of the lack of detectable entry of the hybrid proteins that were constitutively secreted instead. If passive sorting-for-entry were responsible for delivery to granules, the expressed proteins would enter the sorting pathway by bulk flow and be independent of aggregation. If this were the case in our experiments, entrance must have been followed by exit from immature granules as a constitutive-like secretion [35 ], as nonaggregated and aggregated fusion proteins were undetectable in secretory lysosomes. A more likely alternative is that monomeric and aggregated forms of the proteins investigated could have been actively excluded from entry into the sorting pathway. Accordingly, they bypassed the secretory lysosome compartment and were directed promptly to a constitutive, secretory pathway. This is consistent with the previous finding that not all soluble proteins that are imported to Golgi become sorted for storage but instead, become secreted [29 ]. However, the routing for ELA-(FKBP)3 is in contrast to that for ELA alone, as stable expression of ELA in RBL cells resulted in targeting of this protein to the densest, granule-containing fractions and conversion into an enzymatically active form [25 ]. Thus, the ELA-(FKBP)3 chimera is clearly routed differently from ELA alone. This difference may be explained by the elimination of a sorting signal for ELA in the chimera, although evidence for the existence of such a signal is lacking. Additionally, the conformation of the chimera, in contrast to that of ELA, may prevent targeting (see below). Finally, targeting could have been prevented because of the lower charge of the chimera compared with ELA if targeting is dependent on the high cationic charge of ELA. In the latter case, the findings do not rule out the posssibility that some granule proteins could form aggregates during sorting-for-entry, e.g., through ionic interactions with proteoglycans that may contribute to targeting.

Aggregation is not the sole mechanism for sorting-for-entry in hematopoietic cells
Our previous results have suggested that cell-specific sorting is important for protein targeting to secretory lysosomes of hematopoietic cells. Thus, even if hematopoietic cells are specialized in sorting the endogenous proteins for storage, they also have the ability to segregate nonhematopoietic proteins, which are normally secreted into granules upon cDNA expression. For instance, the normally secreted liver proteins lipopolysaccharide-binding protein [36 ] and {alpha}1-microglobulin [37 ] were targeted for granules when expressed in hematopoietic cells [29 , 38 ]. These data demonstrate that the targeting mechanisms for secretory lysosomes are not specific for endogenous granule proteins. The ability to study the reversible aggregation of the chimeric proteins investigated by using cell-permeable ligands gives novel possibilities to explore the role of aggregation. As ELA-(FKBP)3 was not sorted but constitutively secreted, the effect of dimeric ligand-induced aggregation was indeed possible to determine. Therefore, the finding of a lack of sorting-for-entry of aggregated ELA-(FKBP)3 also argues against an aggregation property as the sole mechanism of targeting. The results from experiments with (FKBP*)4-FCS-hGH led to similar conclusions. Thus, in the latter case, reversal of ER retention by monomeric ligand to allow Golgi import did not promote sorting of the fusion protein or aggregates thereof induced by removal of the monomeric ligand. In addition, hGH generated from (FKBP*)4-FCS-hGH by endogenous furin cleavage was also constitutively secreted and not sorted. However, the retention in secretory lysosomes might still be affected by aggregation, as our results primarily reflect a lack of sorting-for-entry in the TGN and not the sorting-by-retention within granules. Sorting in endocrine cells may involve reversible insolubility or aggregation to a granule matrix not investigated here.

Trafficking to secretory lysosomes may take more than one route (see Introduction). The MPR system uses the endosomal route, and a MPR-independent route originates in the dense vesicles within the TGN [14 ]. The two pathways may provide (partly overlapping) sorting for separate regions of the secretory lysosome, a lytic, and a storage region [13 , 14 ]. If two pathways operate normally in trafficking secretory lysosomes, our conclusions on the role of aggregation for sorting are valid with the premise that both pathways are open in the cells we analyzed. An endosomal pathway is necessary to explain the multivesicular body-like morphology of the secretory lysosomes in these cells. We have no proof for a dense vesicle pathway originating in the TGN. To be cautious, our conclusion that aggregation is not the sole mechanism for sorting may be valid only for an endosomal trafficking pathway and not for an alternative pathway if the latter were silent in the cell lines studied.

Aggregation increases cellular retention
We observed that the dimeric ligand promoted the accumulation of ELA-(FKBP)3 upon expression in the hematopoietic cells. Thus, reversible aggregation caused increased cell retention. A fraction of the newly synthesized ELA-(FKBP)3 remained in the ER after a 1-h radiolabel chase, as reflected by the lack of complex glycoforms that are generated only after Golgi import (Fig. 3) . The retention observed may be a result of protection of the aggregates against degradation in the ER. Retention may also be a result of stimulation of biosynthesis by aggregate-dependent inhibition of retrograde movement of nascent polypeptide across the microsomal membrane [39 ]. In addition, the (FKBP*)4-FCS-hGH aggregates were primarily retained in the ER.

Quality control for sorting in the Golgi complex
Aggregates can be of various kinds. Misfolded proteins form aggregates that are eliminated by degradation, and they are therefore actively prevented from sorting and retention. The dense core proteins of the secretory granules of endocrine cells and secretory lysosomes of hematopoietic cells are of a different kind, as they retain a conformation that permits easy dissolution upon discharge. If the ligand-induced aggregates are artificial and different than the aggregates normally generated in cells during granule storage, they may not reflect a normal, aggregation-driven sorting-for-entry if existing. Ligand-regulated aggregation is reversible [23 ] and may therefore be relevant for packaging proteins in an aggregated state for storage and not reflect the aggregation of misfolded proteins. It is however possible that the chimeras and multimers thereof could have improper folding so as to prevent sorting-for-entry. One could speculate that conformation is important for targeting. When truncated proteins are expressed in hematopoietic cell lines, secretion instead of sorting is observed. For instance, deletion of the propeptide rendered truncated promyeloperoxidase susceptible to degradation in the ER, but what was exported to the Golgi was secreted and not retained for storage [40 ]. Similarly, a truncated TNF receptor form that lacked the transmembrane and the cytosol sequences was also not subject to sorting [29 ].

Secretory and membrane proteins acquire native conformations in the ER before final export to Golgi [41 ]. A quality control identifies improperly folded proteins that are diverted to degradation. In addition, there is growing evidence for a secretory pathway quality control operating in the Golgi and the endosomal system [42 ]. This has been observed in yeast, where misfolded proteins are conveyed for vacuolar degradation instead of secretion [43 ]. The observation that the aggregation of furin promoted targeting of this protein for lysosomes supports the existence of a quality control for the disposal of non-native protein in compartments other than the ER [44 ]. The physico-chemical properties of a protein may also affect its fate, sorting or not sorting, in the distal part of the secretory pathway of hematopoietic cells. Our results suggest that truncated and chimeric proteins may be identified by a quality control and directed for constitutive secretion. A Golgi-based quality control would act on any protein to exclude non-native proteins from sorting. However, the hGH released from (FKBP*)4-FCS-hGH by furin cleavage was constitutively secreted and not sorted, as was the intact fusion protein, although the hGH presumably had a native conformation. Furin cleavage to generate native hGH may however have taken place distal to the final sorting station. Thus, ELA was observed to be sorted when expressed in the hematopoietic cell line RBL [25 ]. In addition, at least certain exogenous proteins are accepted by the quality control and sorted for storage when expressed in their native states in hematopoietic cells [29 ]. Conversely, the ELA-(FKBP)3 and (FKBP*)4-FCS-hGH imported to the Golgi seemed to escape quality control and were directed to constitutive secretion, independently of the aggregation state. Our data may suggest constitutive secretion as an alternative to lysosomal degradation, as a mechanism to dispose of improperly folded proteins from the cell.

In conclusion, reversible protein aggregation increased cellular retention but not targeting to secretory lysosomes of hematopoietic cells. Our results indicate that protein multimerization/aggregation per se is not enough to facilitate sorting-for-entry to secretory lysosomes in hematopoietic cells. Targeting may require a proper folding, and non-native proteins may be eliminated from sorting by constitutive secretion as part of a post-ER quality control. Therefore, we speculate on the existence of a distal secretory pathway quality control that excludes improperly folded proteins from sorting.


   ACKNOWLEDGEMENTS
 
The Swedish Cancer Foundation, the Swedish Foundation for Pediatric Cancer, the Alfred Österlund Foundation, and funds from the Lund University Hospital supported this work. We are grateful to Ann-Maj Persson and Marta Sallés Aromir for technical assistance.

Received February 11, 2003; revised June 13, 2003; accepted June 29, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rothman, J. E., Wieland, F. T. (1996) Protein sorting by transport vesicles Science 272,227-234[Abstract]
  2. Arvan, P., Castle, D. (1998) Sorting and storage during secretory granule biogenesis: looking backward and looking forward Biochem. J. 332,593-610
  3. Gorr, S. U., Jain, R. K., Kuehn, U., Joyce, P. B., Cowley, D. J. (2001) Comparative sorting of neuroendocrine secretory proteins: a search for common ground in a mosaic of sorting models and mechanisms Mol. Cell. Endocrinol. 172,1-6[CrossRef][Medline]
  4. Tooze, S. A., Martens, G. J., Huttner, W. B. (2001) Secretory granule biogenesis: rafting to the SNARE Trends Cell Biol. 11,116-122[CrossRef][Medline]
  5. Borregaard, N., Cowland, J. B. (1997) Granules of the human neutrophilic polymorphonuclear leukocyte Blood 89,3503-3521[Free Full Text]
  6. Gullberg, U., Bengtsson, N., Bulow, E., Garwicz, D., Lindmark, A., Olsson, I. (1999) Processing and targeting of granule proteins in human neutrophils J. Immunol. Methods 232,201-210[CrossRef][Medline]
  7. Kornfeld, S., Mellman, I. (1989) The biogenesis of lysosomes Annu. Rev. Cell Biol. 5,483-525[CrossRef][Medline]
  8. Griffiths, G. M., Argon, Y. (1995) Structure and biogenesis of lytic granules Curr. Top. Microbiol. Immunol. 198,39-58[Medline]
  9. Le Cabec, V., Calafat, J., Borregaard, N. (1997) Sorting of the specific granule protein, NGAL, during granulocytic maturation of HL-60 cells Blood 89,2113-2121[Abstract/Free Full Text]
  10. Dannies, P. S. (1999) Protein hormone storage in secretory granules: mechanisms for concentration and sorting Endocr. Rev. 20,3-21[Abstract/Free Full Text]
  11. Dannies, P. S. (2001) Concentrating hormones into secretory granules: layers of control Mol. Cell. Endocrinol. 177,87-93[CrossRef][Medline]
  12. Gerdes, H. H., Rosa, P., Phillips, E., Baeuerle, P. A., Frank, R., Argos, P., Huttner, W. B. (1989) The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calcium-induced aggregation J. Biol. Chem. 264,12009-12015[Abstract/Free Full Text]
  13. Dell’Angelica, E. C., Mullins, C., Caplan, S., Bonifacino, J. S. (2000) Lysosome-related organelles FASEB J. 14,1265-1278[Abstract/Free Full Text]
  14. Blott, E. J., Griffiths, G. M. (2002) Secretory lysosomes Nat. Rev. Mol. Cell Biol. 3,122-131[CrossRef][Medline]
  15. Dahlgren, C., Carlsson, S. R., Karlsson, A., Lundqvist, H., Sjolin, C. (1995) The lysosomal membrane glycoproteins Lamp-1 and Lamp-2 are present in mobilizable organelles, but are absent from the azurophil granules of human neutrophils Biochem. J. 311,667-674
  16. Cieutat, A. M., Lobel, P., August, J. T., Kjeldsen, L., Sengelov, H., Borregaard, N., Bainton, D. F. (1998) Azurophilic granules of human neutrophilic leukocytes are deficient in lysosome-associated membrane proteins but retain the mannose 6-phosphate recognition marker Blood 91,1044-1058[Abstract/Free Full Text]
  17. Cham, B. P., Gerrard, J. M., Bainton, D. F. (1994) Granulophysin is located in the membrane of azurophilic granules in human neutrophils and mobilizes to the plasma membrane following cell stimulation Am. J. Pathol. 144,1369-1380[Abstract]
  18. Burkhardt, J. K., Hester, S., Lapham, C. K., Argon, Y. (1990) The lytic granules of natural killer cells are dual-function organelles combining secretory and pre-lysosomal compartments J. Cell Biol. 111,2327-2340[Abstract/Free Full Text]
  19. Peters, P. J., Borst, J., Oorschot, V., Fukuda, M., Krahenbuhl, O., Tschopp, J., Slot, J. W., Geuze, H. J. (1991) Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes J. Exp. Med. 173,1099-1109[Abstract/Free Full Text]
  20. Dragonetti, A., Baldassarre, M., Castino, R., Demoz, M., Luini, A., Buccione, R., Isidoro, C. (2000) The lysosomal protease cathepsin D is efficiently sorted to and secreted from regulated secretory compartments in the rat basophilic/mast cell line RBL J. Cell Sci. 113,3289-3298[Abstract]
  21. Liu, L., Oren, A., Ganz, T. (1995) Murine 32D c13 cells–a transfectable model of phagocyte granule formation J. Immunol. Methods 181,253-258[CrossRef][Medline]
  22. Seldin, D. C., Adelman, S., Austen, K. F., Stevens, R. L., Hein, A., Caulfield, J. P., Woodbury, R. G. (1985) Homology of the rat basophilic leukemia cell and the rat mucosal mast cell Proc. Natl. Acad. Sci. USA 82,3871-3875[Abstract/Free Full Text]
  23. Rollins, C. T., Rivera, V. M., Woolfson, D. N., Keenan, T., Hatada, M., Adams, S. E., Andrade, L. J., Yaeger, D., van Schravendijk, M. R., Holt, D. A., Gilman, M., Clackson, T. (2000) A ligand-reversible dimerization system for controlling protein-protein interactions Proc. Natl. Acad. Sci. USA 97,7096-7101[Abstract/Free Full Text]
  24. Rivera, V. M., Wang, X., Wardwell, S., Courage, N. L., Volchuk, A., Keenan, T., Holt, D. A., Gilman, M., Orci, L., Cerasoli, F., Jr, Rothman, J. E., Clackson, T. (2000) Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum Science 287,826-830[Abstract/Free Full Text]
  25. Gullberg, U., Lindmark, A., Lindgren, G., Persson, A. M., Nilsson, E., Olsson, I. (1995) Carboxyl-terminal prodomain-deleted human leukocyte elastase and cathepsin G are efficiently targeted to granules and enzymatically activated in the rat basophilic/mast cell line RBL J. Biol. Chem. 270,12912-12918[Abstract/Free Full Text]
  26. Kreider, B. L., Phillips, P. D., Prystowsky, M. B., Shirsat, N., Pierce, J. H., Tushinski, R., Rovera, G. (1990) Induction of the granulocyte-macrophage colony-stimulating factor (CSF) receptor by granulocyte CSF increases the differentiative options of a murine hematopoietic progenitor cell Mol. Cell. Biol. 10,4846-4853[Abstract/Free Full Text]
  27. Lindmark, A., Persson, A. M., Olsson, I. (1990) Biosynthesis and processing of cathepsin G and neutrophil elastase in the leukemic myeloid cell line U-937 Blood 76,2374-2380[Abstract/Free Full Text]
  28. Gullberg, U., Lindmark, A., Nilsson, E., Persson, A. M., Olsson, I. (1994) Processing of human cathepsin G after transfection to the rat basophilic/mast cell tumor line RBL J. Biol. Chem. 269,25219-25225[Abstract/Free Full Text]
  29. Bulow, E., Nauseef, W. M., Goedken, M., McCormick, S., Calafat, J., Gullberg, U., Olsson, I. (2002) Sorting for storage in myeloid cells of nonmyeloid proteins and chimeras with the propeptide of myeloperoxidase precursor J. Leukoc. Biol. 71,279-288[Abstract/Free Full Text]
  30. Schreiber, S. L. (1991) Chemistry and biology of the immunophilins and their immunosuppressive ligands Science 251,283-287[Abstract/Free Full Text]
  31. Clackson, T., Yang, W., Rozamus, L. W., Hatada, M., Amara, J. F., Rollins, C. T., Stevenson, L. F., Magari, S. R., Wood, S. A., Courage, N. L., Lu, X., Cerasoli, F., Jr, Gilman, M., Holt, D. A. (1998) Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity Proc. Natl. Acad. Sci. USA 95,10437-10442[Abstract/Free Full Text]
  32. Denault, J. B., Leduc, R. (1996) Furin/PACE/SPC1: a convertase involved in exocytic and endocytic processing of precursor proteins FEBS Lett. 379,113-116[CrossRef][Medline]
  33. Burkhardt, J. K., Hester, S., Argon, Y. (1989) Two proteins targeted to the same lytic granule compartment undergo very different posttranslational processing Proc. Natl. Acad. Sci. USA 86,7128-7132[Abstract/Free Full Text]
  34. Le Borgne, R., Alconada, A., Bauer, U., Hoflack, B. (1998) The mammalian AP-3 adaptor-like complex mediates the intracellular transport of lysosomal membrane glycoproteins J. Biol. Chem. 273,29451-29461[Abstract/Free Full Text]
  35. Arvan, P., Kuliawat, R., Prabakaran, D., Zavacki, A. M., Elahi, D., Wang, S., Pilkey, D. (1991) Protein discharge from immature secretory granules displays both regulated and constitutive characteristics J. Biol. Chem. 266,14171-14174[Abstract/Free Full Text]
  36. Schumann, R. R., Leong, S. R., Flaggs, G. W., Gray, P. W., Wright, S. D., Mathison, J. C., Tobias, P. S., Ulevitch, R. J. (1990) Structure and function of lipopolysaccharide binding protein Science 249,1429-1431[Abstract/Free Full Text]
  37. Akerstrom, B., Logdberg, L. (1990) An intriguing member of the lipocalin protein family: alpha 1-microglobulin Trends Biochem. Sci. 15,240-243[CrossRef][Medline]
  38. Bulow, E., Gullberg, U., Olsson, I. (2000) Structural requirements for intracellular processing and sorting of bactericidal/permeability-increasing protein (BPI): comparison with lipopolysaccharide-binding protein J. Leukoc. Biol. 68,669-678[Abstract/Free Full Text]
  39. Ooi, C. E., Weiss, J. (1992) Bidirectional movement of a nascent polypeptide across microsomal membranes reveals requirements for vectorial translocation of proteins Cell 71,87-96[CrossRef][Medline]
  40. Andersson, E., Hellman, L., Gullberg, U., Olsson, I. (1998) The role of the propeptide for processing and sorting of human myeloperoxidase J. Biol. Chem. 273,4747-4753[Abstract/Free Full Text]
  41. Ellgaard, L., Molinari, M., Helenius, A. (1999) Setting the standards: quality control in the secretory pathway Science 286,1882-1888[Abstract/Free Full Text]
  42. Arvan, P., Zhao, X., Ramos-Castaneda, J., Chang, A. (2002) Secretory pathway quality control operating in Golgi, plasmalemmal, and endosomal systems Traffic 3,771-780[CrossRef][Medline]
  43. Hong, E., Davidson, A. R., Kaiser, C. A. (1996) A pathway for targeting soluble misfolded proteins to the yeast vacuole J. Cell Biol. 135,623-633[Abstract/Free Full Text]
  44. Wolins, N., Bosshart, H., Kuster, H., Bonifacino, J. S. (1997) Aggregation as a determinant of protein fate in post-Golgi compartments: role of the luminal domain of furin in lysosomal targeting J. Cell Biol. 139,1735-1745[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0203066v1
74/5/800    most recent
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 Google Scholar
Google Scholar
Right arrow Articles by Rosén, H.
Right arrow Articles by Olsson, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rosén, H.
Right arrow Articles by Olsson, I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS