Rab36 regulates the spatial distribution of late endosomes and lysosomes through a similar mechanism to Rab34

Abstract

Small GTPase Rab36 is homologous to Rab34 with 56% amino acid sequence identity. Rab34 was characterized as a Golgi-associated Rab protein and regulates lysosomal positioning through interaction with RILP; however, the properties and functions of Rab36 have not been investigated. To investigate Rab36, we constructed EGFP-Rab36 wild type, the active GTP-bound mutant EGFP-Rab36Q116L and negative GDP-bound mutant EGFP-Rab36T71N. Expression of EGFP-Rab36 wild type revealed that Rab36 co-localized with Golgi markers GM130, Syntaxin 5 and TGN46 in Hela cells, indicating Rab36 is associated with Golgi apparatus. Over-expression of Rab36 induced late endosome and lysosome clustering around the Golgi apparatus, marked by LBPA, CD63, Lamp1 and Lamp2, without effects on early endosomal compartment marked by EEA1. GST-pulldown assay demonstrated that Rab36 can also interact with RILP. In addition, the binding region for Rab36 is in the C-terminal region (aa199-401) of RILP. Our data suggested that Rab36 may regulate the spatial distribution of late endosomes and lysosomes through a similar mechanism to Rab34.

Keywords::

• Rab36
• Rab34
• RILP
• late endosome
• lysosome

Introduction

Late endosome and lysosome are the meeting points for both endocytic protein transport pathway and synthetic protein transport pathway, which are responsible for degradation of cellular wastes and internalized substrates, providing nutrients and energy and sustaining cell signaling [1–3]. Other lysosome-related organelles (LRO) in specialized eukaryotic cells, such as melanosome, lytic granule and platelet-dense granule, play important roles in immuno-defence processes [4]. Defects in the proper function of late endosome and lysosome are related to various lysosomal storage diseases, and give rise to many immunological problems [5]. The distribution and morphology of late endosome/lysosome are closely linked to their physiological functions, and abnormality of morphology and defects in the function of these organelle usually result in diseases [6].

The underlying mechanisms for the regulation of morphology and distribution of late endosome/lysosome were investigated actively in the past decade. Rab5 serves as a membrane organizer in the early endosomal membrane maturation, and over expression of Rab5-GTP induces clustered and enlarged early endosomes [7]. Rab7 is a key molecule in lysosome biogenesis, and Rab7-GTP causes similar effects to Rab5-GTP on lysosomes [8]. The mechanism for Rab7 regulating lysosomal morphogenesis was uncovered through a detailed investigation of Rab7-interacting lysosomal protein (RILP), which regulates lysosomal morphology and distribution through interaction with dyneine/dynactin components [9,10]. Golgi-associated Rab34 shares the same effector RILP as Rab7 to regulate lysosomes peri-Golgi distribution [11,12]. Vem6, a component of HOPs complex supposed as GEF of Rab7, also possesses the property to induce clustering and enlarged late endosome/lysosome, though this regulation seems independent of Rab7 GTPase activity [13].

As described above, Rab proteins play key roles in the regulation of morphology and distribution of late endosome and lysosome. Like other small GTPases, Rab proteins exert their biological functions dependent of GTPase cycle. Rab protein binds to GDI (GDP dissociation inhibitor) as GDP-bound negative form in cytosol. GEF (guanine nucleotide exchange factor) recruits Rab protein to membrane and converts it to GTP-bound active form. GTP-bound Rab proteins interact with diverse downstream effectors to regulate multiple membrane traffic processes, such as vesicle budding, vesicle transport, vesicle docking and fusion, and these processes may regulate the morphology and distribution of cellular organelle [14].

So far, about 70 members of Rab proteins have been identified in mammalian cells, but many of them have not been investigated extensively. In our previous studies, we described that Rab34 regulates lysosomal distribution, and Rab34 is homologous to another Rab protein Rab36 [11]. However, the property and function of Rab36 have not been investigated in detail yet. Here we present comparable data showing that Rab36 regulates the spatial distribution of late endosome and lysosome in a similar way to Rab34.

Materials and methods

Antibodies

The monoclonal antibodies (mAbs) GM130, EEA1 and TGN38 were from BD (BD Biosciences, Palo Alto, CA), mAbs against human Lamp1, CD63 and rat Lamp2 were obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa (Department of Biological Science, Iowa City, IA, USA). mAb against Golgi mannosidase II was purchased from Babco (Berkeley, CA, USA). Syntanxin5 antibody was described [15]. mAb against Myc-tag (9E10) was obtained from American Type Culture Collection (ATCC, Manassas, VA 20108, USA). mAb against EGFP were purchased from the Clontech Laboratories, BD Biosciences (Palo Alto, CA, USA). mAb against lysobisphosphatidic acid (LBPA) was a generous gift from Dr Jean Gruenberg (University of Geneva, Switzerland). HRP-conjugated secondary antibodies were purchased from Pierce (Rockford, IL, USA). Texas red-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA).

Expression constructs

The coding region human Rab36 was retrieved from cDNA library of human fetal brain (BD Clontech) by PCR with primer 1 (5′-AGA TCT CGA GTG AGG TCC TCC CTG ACA CCT TTG-3′) and 2 (5′-AA GGA TCC TTA GCA GCA GCC CAG GCT GGA GGG-3′), and cloned into Xho I/BamH I sites of pEGFP-C1 vector (BD Clontech) to generate construct for expression of GFP-Rab36WT in mammalian cells, or subcloned into BamH I/Xho I sites of pGEX-4T-1 vector (Amersham Biosciences, Arlington Heights, IL, USA) to express GST-Rab36WT protein. Rab36T71N and Rab36Q116L mutants were prepared by using the standard PCR mutagenesis methods. All constructs were confirmed by DNA sequencing. pDmyc-RILP, pEGFP-Rab34WT, GST-Rab34WT, GST-RILP, myc-RILP(1-198), myc-RILP(199-401) were described previously [12].

Tissue expression assessed by cDNA panel

Human multiple tissues cDNA panels (Clontech Laboratories, BD Biosciences, Palo Alto, CA, USA) were used for PCR-based analysis for the transcripts of Rab36 and Rab34. Primer 3 (5′ ATG AGG TCC TCC CTG ACA CCT TTG 3′) and primer 4 (5′ TTA GCA GCA GCC CAG GCT GGA GGG 3′) were used to amplify Rab36. Primer 5 (5′ ATG AAC ATT CTG GCA CCC GTG CGG 3′) and primer 6 (5′ TCA TGG GCA ACA TGT GGG CTT CTT 3′) were used to amplify Rab34. Primer 7 (5′ TGA AGG TCG GAG TCA ACG GAT TTG GT 3′) and primer 8 (5′ CAT GTGGGC CAT GAG GTC CAC CAC 3′) were used to amplify a control cDNA fragment of G3PDH. The PCR products were resolved by agarose gel electrophoresis.

Cell culture, transfection and immunofluorescence microscopy

NRK and HeLa cells were grown in RPMI 1640 or DMEM media at 37°C in an incubator with 5% CO₂. Transfection of plasmids was carried out by using lipofectamine 2000 reagents (Invitrogen, Gaithersburg, MD, USA) according to the manufacturer's protocols. 18–20 hours after transfection, the cells were fixed and processed for immunofluorescence labeling with the indicated markers. Nocodazole (Sigma-Aldrich, St Louis MO, USA) treatment and the immunofluorescence experiments were performed as described [11]. Confocal microscopy was performed with Carl Zeiss LSM5 EXITER laser scanning microscope (Zeiss, Jena, Germany).

GST-pulldown assay

GST-pulldown assay was performed as described [11]. Briefly, the transfected cells were harvested and lysed in binding buffer (containing 20 mM HEPES, pH7.4, 100mM NaCl, 5 mM MgCl₂, 1% TX-100, and EDTA-free proteinase inhibitor cocktail [Roche, Nutley, NJ, USA]). A total of 25 μg GST-fusion proteins was coupled to the GST-Sepharose 4B resin and incubated with cell lysates. After extensively wash, the bound proteins were resolved by SDS-PAGE and transferred to nitrocellulose filter. The filter was blocked with 5% milk in PBS and then incubated with primary antibody for 1 h at room temperature, followed by HRP-conjugated secondary antibody for 1 h at room temperature. The blots were detected using ECL system (Pierce, Rockford, IL, USA).

Results

Analysis of sequence and examination of tissue expression of Rab36 transcript

Mori and colleagues firstly isolated human Rab36 gene from the homozygous deletions in chromosome 22q11.2 responsible for malignant rhabdoid tumors (MRTs), and characterized the open reading frame encoding Rab36 protein of 333 amino acids [16]. When comparing Rab36 amino acids sequence from divergent organism sources using BLAST tool, we found that the N-terminal 66aa extension in human Rab36 was not conserved among divergent organisms (Figure 1A), since no homologous residues was found in the N-terminal flanking regions of Rab36 from other organism except Homos sapiens, thus we propose that the amino acid sequence of human Rab36 protein in full length must be 267aa harbouring all conserve motifs for small GTPase.

Figure 1.  (A) Amino acids sequence alignment analysis of Rab36 from several organisms, showing the N-terminal 66aa extension in human Rab36 was not conserved among divergent organisms. (B) Comparison of the amino acids sequences of Rab36 and Rab34, residues identical were shown in black background. (C) Examination of tissue expression of transcripts of human Rab36 and Rab34 by PCR using multiple tissue cDNA panels, indicating Rab36 and Rab34 have similar tissue expression pattern.

Sequence alignment showed that Rab36 protein share high homology with Rab34 with 56% amino acids identity (Figure 1B), suggesting that these two proteins may be expressed in different human tissues with similar biological functions. We examined the expression levels of Rab36 and Rab34 transcripts by PCR with specific primers, using human multiple tissue cDNA panels which contains eight different tissues indicated in Figure 1C. Unexpectedly, the data showed that Rab36 was expressed ubiquitously in all tissues, just similar to Rab34, though the cDNA level of Rab36 was lower than Rab34.

Rab36 is associated with Golgi apparatus

To examine the subcellular localization of Rab36, the cDNA retrieved from human fetal brain cDNA library encoding Rab36 of 267aa was subcloned into pEGFP-C1 vector. The expression construct was transfected into HeLa cells. The localization of the expressed GFP-Rab36 was studied by immuno-labeling antibodies against Golgi markers (Figure 2). As shown in Figure 2, although a fraction of GFP-Rab36 presents in cytosol, the membrane-bound GFP-Rab36 are associated with Golgi apparatus marked by GM130, Syntaxin5 and TGN46 in HeLa cells. Further examination indicated that Rab36 is preferentially associated at trans-Golgi network, as the GFP-Rab36-associated membrane is partially segregated from cis-Golgi cisternae marked by GM130, but well co-localized with trans-Golgi apparatus marked by TGN46 (magnified panels, Figure 2). The Golgi-association of Rab36 was verified by co-localization with GM130, Man II and TGN38 in NRK cells (data not shown). The results demonstrated that Rab36 and Rab34 have similar subcellular location. Our data also suggested that the N-terminal 66aa extension in human Rab36 characterized by Mori et al. is not necessary for its membrane location.

Figure 2.  Rab36 is associated with Golgi apparatus. HeLa cells were transfected with EGPF-Rab36WT, and processed for immuno-labeling with antibodies against GM130 to label cis-Golgi, syntaxin 5 to label medium Golgi or TGN46 to label trans-Golgi network. The right panels were magnified to show the co-localization of Rab36 with Golgi apparatus, and demonstrated that Rab36 is preferentially associated with TGN. Bar = 20 μm. This Figure is reproduced in colour in the online version of Molecular Membrane Biology.

Rab36 regulates the spatial distribution of late endosomes and lysosomes

The previous works by Wang and Hong indicated that Golgi-associated Rab34 regulated lysosomal distribution [11]. Because Rab36 shares high amino acid sequence identity with Rab34, Rab36 may possess similar functions to Rab34. To verify this hypothesis, we expressed GFP-Rab36WT in HeLa cells, and examined the effects of over-expressing Rab36 on endocytic compartments. After over-expressing EGFP-Rab36WT, HeLa cells were immuno-labeled by EEA1, LBPA (lysobisphosphatidic acid, unique lipid in MVB [multi-vesicular body]), CD63 and Lamp1. It was observed that the late endocytic compartments: late endosomes and lysosomes were redistributed to the peri-nuclear region by Rab36, but the size of the vesicles was not altered (Figure 3A). The relocated vesicles were not overlapped with Golgi apparatus marked by GFP-Rab36 (the inset in Figure 3A). The distribution of the early endocytic compartments marked by EEA1 were not altered essentially (upper panel in Figure 3A), indicating the effects of over-expressing Rab36 on late endosomes and lysosomes are specific.

Figure 3.  Rab36 regulates peri-nuclear distribution of late endosomes and lysosomes from peripheries. (A) HeLa cells were transfected with EGFP-Rab36WT, and processed for immuno-labeling with antibodies against EEA1, LBPA, CD63 and Lamp1 respectively, showing overexpression of Rab36 induced peri-nuclear distribution of late endosomes and lysosomes marked by LBPA, CD63 or Lamp1, but had no effects on early endosomes marked by EEA1. The inset showed the clustered vesicles not overlapping with Rab36-marked Golgi apparatus. (B) Expression of active mutant form (Q116L) but not negative mutant form (T71N) of Rab36 redistributes late endocytic compartments to peri-nuclear region. (C) The effects of Rab36 on the distribution of late endosomes and lysosomes were further confirmed by over-expressing EGFP-Rab36WT in NRK cells and examined with antibodies against LBPA or Lamp2. The effects of over-expressing Rab34 were also compared. (D) NRK cells expressing EGFP-Rab36WT were treated with nocodazole of 10 μg/ml for 1 hour at 37°C, then processed for immuno-labeling with anti-TGN38 or anti-Lamp2. The result showed that GFP-Rab36 is redistributed to Golgi-fragment, and the late endosomes/lysosomes are dispersed. Bar = 20 μm. This Figure is reproduced in colour in the online version of Molecular Membrane Biology.

To see whether Rab36's effects on late endosomal/lysosomal distribution is dependent of guanine nucleotides binding activities, we generated Rab36Q116L and Rab36T71N mutant forms. Rab36Q116L lacks GTPase activity and is referred to as constitutive active form, while Rab36T71N prefer binding GDP and is referred to as dominant negative form. The over-expression results showed that Rab36Q116L is associated with the Golgi apparatus (marked by TGN46, data not shown) like wild type of Rab36, while Rab36T71N is cytosolic and distributes throughout cytoplasm. As shown in Figure 3B, lysosomal distribution is altered significantly by Rab36Q116L, but not Rab36T71N, suggesting Rab36 regulates the distribution of late endosomes and lysosomes dependent of guanine nucleotides binding activities.

The comparable experiments were carried out in NRK cells. To further confirm the effects of Rab36, EGFP-Rab36WT was expressed in NRK cells, and the distribution of late endosomes and lysosomes were examined by immuno-labeling LBPA and Lamp2, respectively. The observations demonstrated that both late endosomes and lysosomes were induced to cluster around peri-nuclear region (Figure 3C). Compared with Rab34, over 90% cells expressing GFP-Rab36 induced late endosomal/lysosomal redistribution, equivalent to the effects of Rab34. The data suggested Rab36 has the same functional capability as Rab34 to regulate the distribution of late endosome and lysosome.

Nocodazole treatment promotes depolymerization of microtubule, resulting in fragmentation of the Golgi apparatus. It was observed that GFP-Rab36 is redistributed to TGN38-labeled Golgi fragment in NRK cells, further confirming that Rab36 is associated with Golgi apparatus (upper panel in Figure 3D). As expected, Lamp2-labeled late endosomes/lysosomes, supposed to be peri-nuclear positioning before treatment, are dispersed randomly by nocodazole treatment (lower panel in Figure 3D), suggesting that the effect of Rab36 on the distribution of late endosomes/lysosomes is dependent of intact microtubule network.

Rab36 interacts with RILP

The previous investigation on Rab34 unveiled a novel mechanism of inter-organellar regulation of lysosome positioning, in that Golgi associated Rab34 regulated lysosome distribution through interaction with RILP (Rab7-interacting lysosomal protein) [11]. To study whether Rab36 regulates the spatial distribution of late endosome and lysosome through a similar mechanism to Rab34, we investigated the interaction between Rab36 and RILP by applying GST-pulldown assay. The in vitro binding assay was processed by using immobilized GST-RILP to incubate with cell lysates containing expressed Rab proteins. As shown in Figure 4A, GST-RILP significantly bound to GFP-Rab36 equivalent to the amount of GFP-Rab34. Further experiments indicated that the interaction of Rab36 with RILP depends on specific guanine nucleotide-binding activity of Rab36, because the GTP-bound Rab36Q116L mutant can bind to RILP, while GDP-bound Rab36T71N inhibited the interaction significantly (Figure 4A). This result indicated RILP is also a down stream effector for Rab36. In addition, we used immobilized GST-Rab36WT to bind RILP from the cell lysates containing expressed myc-RILP, myc-RILP(1-198) and myc-RILP(199-401), respectively, and the results further confirmed the interaction between Rab36 and RILP, and identified the C-terminal region of RILP responsible for the interaction with Rab36 or Rab34 (Figure 4B). Taken together, Rab36 may employ a similar mechanism to Rab34 for regulating the spatial distribution of late endosome and lysosome through interaction with RILP.

Figure 4.  Rab36 interacts with RILP. (A) GST-RILP was immobilized on GST-sepharose beads and used to pull down GFP-Rab proteins from cell lysates containing expressed GFP-Rab36 or Rab34. The results showed RILP specifically interacts with Rab34, Rab36WT or Rab36Q116L, but weakly interacts with Rab36T71N. GST was used for control. (B) Characterization of the region in RILP interacting with Rab36. GST-Rab proteins were immobilized on GST-sepharose beads and used to pull down myc-tagged proteins from cell lysates containing expressed myc-RILP, myc-RILP(1–198) or myc-RILP (199–401). The data further confirmed the interaction between Rab36 and RILP, and indicated both Rab36 and Rab34 interact with C-terminal region of RILP.

Discussion

Rab proteins represent the largest family in the Ras superfamily. So far about 70 members of Rab proteins have been identified, but many of them have not been investigated in detail, and their accurate biological functions remain to be elucidated extensively. Rab36 and Rab34 share high homology and both are associated with Golgi apparatus [11,16]. However, the cell biological functions of Rab36 have not been studied. Our present data showed that Rab36 is preferentially associated with Trans-Golgi network (TGN). In addition, Rab36 regulates the late endosome and lysosome redistribute to peri-nuclear region from peripheries. Previous studies indicated that Rab7 and Rab34 regulate lysosomal distribution and morphogenesis through interacting with RILP [10,11], which mechanically orchestrates vesicles moving from peripheries to MTOC (microtubule organizing center) along microtubule through interacting with dynein/dynactin complex [10]. Our further experiments demonstrated that Rab36 interacts with RILP, suggesting that RILP is also a down stream effector for Rab36, and Rab36 may regulate the distribution of late endosome and lysosome in a similar mechanism to Rab34 through interaction with RILP. Thus we presented complemental and significant knowledge to further understand the mechanisms for the regulation of late endosome/lysosomal positioning, and especially for interorganellar regulation of organelle positioning.

Besides Rab36, several molecules have been discovered to regulate the morphogenesis of late endocytic compartments, such as Rab34, Rab7, RILP and Vam6 [9–11,13]. Rab36, Rab34 and Rab7 interact with RILP, while vam6 is homolog to yeast VPS39p, one subunit of HOPs complex (homotypic fusion and vacuole protein sorting complex) and serves as GEF (guanine nucleotide exchange factor) for Rab7 [17,18]. Rab36/Rab34-RILP complex regulates peri-nuclear distribution of the late endosome and lysosome, without affecting the sizes of organelle. Rab7-RILP complex regulates not only the peri-nuclear distribution but also membrane fission and fusion of the late endocytic compartments, resulting in enlarged vesicles. Vam6 possesses similar activities to Rab7 and RILP. The above results demonstrated that Rab7-RILP interaction may play a central role in the regulation of late endosomal/lysosomal morphogenesis, and hypothetically Rab36 and Rab34 play roles in cis-regulations, and Rab7, RILP and Vam6 play roles in trans-regulations.

Rab proteins and their effectors are the major actors in the mechanism of protein-trafficking disorders. Dysfunctions of Rab proteins are associated with many diseases [19–21]. Such as, mutation of Rab7 is related to Charcot-Marie-Tooth type 2B disease [22], and mutation of Rab27A is associated to Griscelli syndrome [23]. Recent investigations indicated that some Rab proteins participate in the regulation of tumor development. Rab25 determines the aggression of breast cancer cell [24], and Rab7 regulates the apoptosis of cancer cells [25]. Because Rab36 was isolated from the ‘critical region’ of homozygous deletions at chromosome 22q11.2, which causes malignant rhabdoid tumors (MRTs) [16,26], Rab36 was supposed to be a tumor suppressor gene. But over-expression of Rab36 did not affect the anchorage-dependent growth of MRT cells [16] suggesting Rab36 may play other pathological function than tumor suppressor factor. The alterations of morphogenesis of lysosomes and lysosome-related organelle (LRO) are closely related to human diseases [4,5]. For example, defect in melanosome results in abnormal pigmentation [27], and aberrant morphology of lysosomes is the phenotype of CHS (Chediak-Higashi syndrome) [28]. Whether the effects of Rab36 on the distribution of late endosome and lysosome are related to the pathological functions deserves further investigations.

Acknowledgements

This work was supported by the start-up fund for new investigators from Xiamen University, China, grant No. 1280X12104.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.