Interferon-gamma selectively induces Rab5a synthesis and processing in mononuclear cells.

Macrophage activation by interferon (IFN)-gamma is characterized by enhanced phagocytosis and killing of internalized pathogens. We studied the effects of IFN-gamma on Rab5a, a GTPase involved in both endocytosis and phagocytosis. IFN-gamma induced the synthesis of Rab5a in mononuclear cells as detected by immunoprecipitation and by Western blotting. Rab5a messenger RNA levels were also increased. Elevated protein expression was detected as early as 6 h following IFN-gamma and was maximal at 24 h. Following IFN-gamma, membrane association of Rab5a:GTP was substantially increased. Rab5b and Rab5c, as well as Rab7 and Rab11, Rab GTPases localized in the endosomal-lysosomal pathway, were unaffected by IFN-gamma. Moreover, Rab5a expression in non-macrophages was unaltered by IFN-gamma. Rab5a is a prenylated protein, and newly synthesized Rab5a was rapidly processed following IFN-gamma. However, elevated geranylgeranylation was not Rab5a-specific since all the Rab5 isoforms were more rapidly prenylated in vitro using cytosol from IFN-gamma-treated cells. Last, guanine nucleotide exchange on Rab5a was elevated about 3-fold in the presence of cytosol from IFN-gamma-treated cells. The selective effect of IFN-gamma on Rab5a, synthesis, processing, and nucleotide exchange suggests that Rab isoforms have closely associated but not identical functions and that selective enhancement of membrane trafficking may play a key role in intracellular killing.

Macrophages (Ms) 1 occupy a central position in the immune system because of their phagocytic and antimicrobial capabilities. As a component of both acquired and innate pathways, Ms respond to lymphokine treatment by acquiring new functions. The activating effects of IFN-␥ on macrophage function are well known (1) and include enhanced intracellular killing of pathogens via phagocytosis (2,3). Similar to endocytosis, phag-ocytosis is regulated by the GTPase Rab5 (4 -6). The Rab5 cycle is well described and involves Rab5 recruitment to membrane sites from a cytoplasmic pool where Rab5 is presumably bound to GDI. Prenylation via mono-and di-geranylgeranylation is essential for binding of Rab5 to intracellular membranes (7,8). Rab5 is active in the GTP form, and membrane association is associated with GDP/GTP exchange via the Rab5 exchange factor (GEF) (4,6,9,10). GTPase hydrolysis terminates its activity and is thought to permit release of Rab5 from the membrane. At least one Rab5 GTPase activating protein (GAP) has been described that putatively acts on membrane-bound Rab5 to enhance GTP-hydrolysis (11). Rab5 is expressed as three isoforms, Rab5a, Rab5b, and Rab5c (12). Rab5 isoform messengers are ubiquitously expressed, and the three proteins have overlapping intracellular distributions. When overexpressed in living cells, all Rab5 isoforms activate endocytosis (12).
Here we report on a highly specific effect of IFN-␥ on Rab5a synthesis, processing, and guanine nucleotide exchange. These findings have important implications in defining the boundaries between the endocytic and phagocytic pathways and on the mechanism of enhanced phagosome maturation following IFN-␥ treatment.
Immunoprecipitation and Western Blots-Cells were lysed with PBS containing 1% Triton X-100, 2 mM EDTA, 10 nM ␣-PMSF, 10 g/ml leupeptin, 10 nM pepstatin-A, 10 g/ml aprotinin, 5 mM NaVO 4 , and 1 mM NaF followed by ultracentrifugation at 100,000 ϫ g, for 45 min at 4°C. For immunoprecipitation, lysates were precleared overnight at 4°C by incubation with 20 l of a 50% slurry of protein-A-Sepharose beads. The supernatants were then incubated with antibodies for 4 -5 h at 4°C, and at 1 h prior to washing, 20 l of a 50% slurry of protein A-Sepharose was added. Beads were washed with lysis buffer, 0.5% sodium deoxycholate; lysis buffer, 0.5% sodium deoxycholate; 500 mM NaCl and 1/20 lysis buffer, 0.5% sodium deoxycholate. Elution was performed in 1ϫ Laemmli sample buffer for 1 h at room temperature. For Western blots, 30 g of total protein (measured by the BCA method, Pierce Chemicals, IL) was loaded per lane, separated by SDS-PAGE, and transferred onto nitrocellulose membranes. Membranes were blocked in PBS, 5% nondairy milk for at least 2 h at room temperature. Primary antibodies were incubated overnight at 4°C and washed with PBS, 0.05% Tween 20. Secondary antibodies were incubated for 45 min at room temperature using a 1:10,000 dilution.
Limiting Dilution RT-PCR-HMs at 5 ϫ 10 6 /ml were treated with hIFN-␥ (100 units/ml) for different periods of time (0, 1, 2, 8, and 18 h). Total RNA was isolated from the Ms by the phenol/guanidine thiocyanate procedure using TRI-Reagent (Molecular Research Center, OH). Complementary DNA was synthesized with a Perkin-Elmer kit using the protocol of the manufacturer. Briefly, 5 g of total RNA in RNase-DNase free autoclaved water was mixed with 3 l of oligo(dT) 15 primer, heated for 15 min at 65°C. After cooling on ice, the following reagents were added: 4 l of 5ϫ first strand buffer, 2 l of 0.1 M dithiothreitol (DTT), 1 l of reverse transcriptase (20 units/ml), 1 l of RNase inhibitor (20 units/ml), 2 l of dNTPs (30 mM each). The mixture was incubated for 90 min at 37°C, heat-inactivated for 5 min at 95°C, and * This work was supported by National Institutes of Health grants (to P. D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡Recipient of a Postdoctoral Fellowship from the Formación de Personal Investigador, Ministerio de Educación y Ciencia, Madrid, Spain.
Prenylation of Fusion Proteins-Prenylation was performed as described previously (4). 8 g of GST-Rab5a, GST-Rab5b, or GST-Rab5c were incubated in 40 l of 50 mM Hepes/KOH, pH 7, 5 mM MgCl 2 , 0.5 mM Nonidet-P40, 1 mM DTT containing 12 M [ 3 H]GGPP for 30 min in the presence of control cytosol or cytosol obtained from HMs treated with IFN-␥ (150 units/ml for 18 h). Reaction was stopped by adding 4ϫ SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE, and the dried gels were exposed to film for 4 days to quantitate incorporated radiolabel.
GDP/GTP Exchange-Protocol was performed as described (16). Briefly, 8 g of GST-Rab5a was incubated with 10 M [ 3 H]GDP for 20 min at 37°C in HBE-loading buffer (HBE containing 1 mM DTT, 100 mM NaCl, and 40 g/ml BSA). A 10-fold excess of HBE-exchange buffer (HBE-loading buffer containing 1 mM GTP-15 mM MgCl 2 ) was added together with 5 l of cytosol (Sephadex G-25 filtered; 0.5 mg/ml) obtained from control macrophages or cytosol from HMs treated with IFN-␥ (18 h, 150 units/ml). GDP/GTP exchange was determined at RT by removing 5-l aliquots at different times (0, 5, 15, and 30 min) and passing the samples through nitrocellulose filter disks coupled to a vacuum system. Filters were washed twice in ice-cold HBE-washing buffer (HBE containing 100 mM NaCl and 10 mM MgCl 2 ) and dried. Radioactivity was determined in a ␤-counter.

RESULTS AND DISCUSSION
IFN-␥ induces the synthesis of a wide variety of proteins in mononuclear cells leading to activation and enhanced intracellular killing (1). Because Rab GTPases are essential for membrane trafficking events leading to phagosome lysosome fusion, we examined the effect of IFN-␥ on intracellular levels of Rab5. Incubation of human macrophages (HMs) with IFN-␥ (100 -200 units/ml) increased both newly synthesized Rab5a protein and Rab5a mRNA levels (Fig. 1). Newly synthesized Rab5a (Fig. 1, panel A) was detected after metabolic labeling and immunoprecipitation with a Rab5a-specific monoclonal anti-

IFN-␥ Induces Rab5a
Synthesis 33902 body 4F11 (6,14), and mRNA levels were recorded by limiting dilution RT-PCR using primers for hRab5a (Fig. 1B). ␤-Actin message levels remained unaltered following IFN-␥. Induction of Rab5a mRNA was detected 1 h after IFN-␥, whereas the increase in Rab5a protein synthesis was first observed after 2 h. Rab5a mRNA levels remained higher in cells treated with IFN-␥ for up to 8 h. After 18 h, Rab5a mRNA levels returned to a basal state approximating that found in untreated cells. Enhanced biosynthesis of Rab5a remained high for at least 18 h. The lack of coincidence of apparent mRNA synthesis and Rab5a protein synthesis may be because of the combination of a rapidly responsive but short lived RNA and a long lived protein. Interferon-␥ is known to regulate a number of rapidly responsive genes. The experiments also revealed that, in addition to enhancing the synthesis of Rab5a (5-8-fold), IFN-␥ had a pronounced effect on the post-translational processing of the protein. A shift in the migration of newly synthesized Rab5a was observed following SDS-PAGE of immunoprecipitated Rab5a. This shift was discernible after 4 h of IFN-␥ treatment and was clearly evident after 6 -8 h. Rapid post-translational processing of Rab5a in untreated macrophages was not detected. Rab5a has two potential geranylation sites at the C terminus of the molecule. Because Rab5a is prenylated in both control and treated macrophages, the rapid shift in migration of Rab5a, as detected by SDS-PAGE following IFN-␥ treatment, may be because of the addition of two prenyl groups. Previous reports showed that following transient expression of Rab5a, the newly synthesized ϳ25-kDa polypeptide is processed by post-translational modification via C-terminal isoprenylation, resulting in a decreased molecular mass (ϳ23 kDa) (7,8). To demonstrate the effect of IFN-␥ on Rab5a synthesis in the absence of processing, we inhibited geranylgeranylation with lovastatin (10 M), a specific inhibitor of isoprenylation (7) (Fig. 1C). Lovastatin had no effect on IFN-␥ induction of Rab5a synthesis. However, compared with cells not exposed to the inhibitor, the processing of Rab5a to an apparent lower molecular mass form was prevented. These data suggest that processing events leading to a mature form of Rab5a are either because of prenylation or occur after prenylation. They also suggested that IFN-␥ signaling results in enhanced prenylation of Rab5a. This conclusion was confirmed by examining the prenylation of the GST-Rab5a fusion protein in the presence of cytosol obtained from control HMs or cells treated with IFN-␥ (18 h, 150 units/ml). As shown in Fig. 1D, there is a substantial increase in GST-Rab5a prenylation in the presence of cytosol from IFN-␥-treated HMs.
Rab5 is expressed as three isoforms. The IFN-␥ effect on Rab5 synthesis was highly specific for Rab5a since none of the other Rab5 isoforms were affected by IFN-␥ treatment either at the mRNA level (Fig. 1B) or at the protein level ( Fig. 2A). RT-PCRs with primers specific for each isoform (i.e. primers that fail to amplify the other isoforms (data not shown)), showed that mRNA levels of Rab5b and Rab5c remain unaltered by IFN-␥ treatment (Fig. 1B). Metabolic labeling of HMs and immunoprecipitation with specific antibodies that exclusively recognize a single isoform (12) showed that IFN-␥ treatment had no effect on Rab5b or Rab5c ( Fig. 2A) synthesis. However, cytosol from cells treated with IFN-␥, when incubated with GST-Rab5b or GST-Rab5c (Fig. 1D), resulted in elevated levels of prenylation on all three Rab5 isoforms. These data indicate that the increase in prenylation is not specific for   FIG. 3. Rab5a function is affected by IFN-␥. Modulation of intracellular location and GDP/GTP exchange rate. A, HMs were treated for different times with hIFN-␥ (150 units/ml) and metabolically labeled as in Fig. 1. Cells were homogenized in HBE buffer, centrifuged to remove nuclei and mitochondria. The post-nuclear supernatants were subjected to differential centrifugation by layering on an 8.5% sucrose cushion and spun at 100,000 ϫ g for 60 min to obtain membranes (m) (pellets after centrifugation) and cytosolic fractions (c) (supernatants after centrifugation). Immunoprecipitation was performed with 4F11 antibody in the presence of detergent as described under "Experimental Procedures." To quantify m/c ratios, radioactivity associated with proteins eluted from protein A-Sepharose beads was counted in a ␤-counter, and the m/c ratios were calculated for each condition. Actual data collected were as follows: Cells were infected with LM hly-. Samples labeled as membranes correspond to total phagosomal membranes from isolated phagosomes (6, 17) (see "Experimental Procedures" for protocol) from cells treated with IFN-␥ (ϩIFN) or nontreated (ϪIFN). Rab5a was immunoprecipitated on ice for 10 min using 4F11 antibody, and eluted nucleotides were separated by thin layer chromatography. GTP:GDP ratio was determined as described (21). A phosphoimager was used to determine GTP:GDP ratios, taking into account that the specific activity of [ 32 P]GDP is two-thirds that of the Rab5a isoform. It is interesting to note that Rab5a appears to be a significantly better substrate for the induced prenyltransferase activity compared with Rab5b or Rab5c. It is possible that other Rab5a-specific factors are induced that allow Rab5a to be more rapidly prenylated. Rab7 and Rab11, GT-Pases acting downstream of Rab5 and localized in the endosomal-lysosomal pathway (18 -20), were not induced by IFN-␥ treatment as shown by Western blot assays of whole cell lysates (Fig. 2B). Interestingly, nonphagocytic cell types such as A431 human epithelial cells, fibroblasts (L929 mouse cell line), or B cells (mouse A20 cell line), which express IFN-␥ receptors, were unaffected (Fig. 2C) (1). However, in macrophage-cell lines such as J774E clone, IFN-␥ clearly induced the synthesis of Rab5a.
In Ms, the effects of IFN-␥ are principally associated with clearance of intracellular pathogens (1,3). Membrane trafficking pathways leading to phagosome-lysosome fusion would be obvious targets of IFN-␥ action (2,3). To delineate the effects of IFN-␥ on Rab5a function, we examined the intracellular localization of Rab5a following IFN-␥ treatment. Metabolically labeled HMs were homogenized in HBE (250 mM sucrose, 0.5 mM EGTA, 20 mM Hepes-KOH, pH 7.2) and fractionated by sedimentation. Rab5a was then immunoprecipitated from membrane and cytosol fractions. Fig. 3A shows that most of the newly synthesized Rab5a following IFN-␥ induction is localized to the membrane fraction (m). Indeed, the membrane/cytosol (m/c) ratio of immunoprecipitated Rab5a after IFN-␥ increased from four in untreated macrophages to nine in IFN-␥-treated cells. Increased membrane association may be because of accelerated geranylgeranylation of newly synthesized Rab5a as shown in Fig. 1D. Taken together these data demonstrate that IFN-␥ specifically enhances Rab5a synthesis and processing (i.e. isoprenylation) in mononuclear cells, including translocation of newly synthesized Rab5a to intracellular membranes. Similar to most low molecular weight GTPases, Rab5 is active in the GTP form (4, 6, 10). As a first approximation of the effects of IFN-␥ on Rab5 function, we examined the guanine nucleotide status of Rab5a on phagosomal membranes. HMs were incubated overnight with [ 32 P]orthophosphate. The cells were then pulsed for 10 min with L. monocytogenes, an intracellular pathogen efficiently internalized by Ms. Listeria containing phagosomes were isolated by differential sedimentation (6,17). Rab5a was rapidly immunoprecipitated following solubilization of the phagosomal membranes. [ 32 P]guanine nucleotides were released from the immunoprecipitates and were separated by thin layer chromatography to allow estimation of the GTP:GDP ratio. As shown in Fig. 3B, the GTP:GDP ratio of membrane-bound Rab5a increased by 2-fold in response to IFN-␥ (from 0.83 to 1.66). We conclude that IFN-␥ not only induces Rab5a expression and processing but also guanine nucleotide exchange or hydrolysis or both.
The GEFs that mediate exchange of GDP for GTP (9) and GAP that promotes the GTP hydrolysis (11) are two key activities that regulate the Rab5 GDP/GTP cycle. Membrane ruffling caused by guanine nucleotide exchange on Rho GTPases has recently been shown as the target of a factor produced by Salmonella typhimurium (16). To examine the effects of IFN-␥ on guanine nucleotide exchange on Rab5a, we used a guanine nucleotide release assay employed by Hardt et al. (16). GST-Rab5a was preloaded with [ 3 H]GDP by incubating the protein and [ 3 H]GDP in the presence of low levels of Mg 2ϩ . Pre-loaded Rab5a was then incubated with cytosol from control cells and from cells pretreated with IFN-␥ (18 h, 150 units/ml). Increased levels of Mg 2ϩ and GTP were added, and the release of [ 3 H]GDP was monitored. Analysis of the GDP/GTP exchange rate showed that the cytosol obtained from HMs treated with IFN-␥ specifically increased the intrinsic GDP/GTP exchange rate of GST-Rab5a (Fig. 3C) by a factor of 3 compared with the rate observed in the presence of control cytosol. It is not clear from our studies how IFN-␥ enhances nucleotide exchange on Rab5. Rabex-5 has been shown to possess Rab5 guanine nucleotide exchange activity. This protein may account for the enhanced activity observed in IFN-␥-treated cytosol (21).
In summary, IFN-␥ induces Rab5a and at least two activities that are coupled to Rab5a function, Rab5a prenylation activity and Rab5a guanine nucleotide exchange activity. Enhanced prenylation activity is clearly not Rab5a-selective although Rab5a is a superior substrate for prenylation following IFN-␥ treatment. Prenyltransferases and molecules that interact exclusively with Rab5 (e.g. GEF or GAP) may be responsible for the selective retention of Rab5a in the membrane fraction. We speculate that enhanced Rab5a levels and function accelerates the rate of phagosome maturation and intracellular killing that accompanies IFN-␥ treatment.
The selective effect of IFN-␥ on Rab5a suggests that each Rab5 isoform regulates a different function along the phagocytic-endocytic pathway. Subdomains of the endocytic-phagocytic pathway were first suggested by the discovery and localization of different Rab GTPases. Our study suggests that this concept can now be extended to a newer level of refinement. Moreover, the results presented in this study constitute the first report that small GTPase action can be modulated by lymphokine signaling.