TEMP was originally discovered as part of RIKEN’s FANTOM3 project [8] as a predicted open-reading frame of 111 amino acids in the murine cDNA clone 2610528J11. Bioinformatic analysis identified that TEMP does not have any significant relationship to other proteins domains or families. However, TEMP is predicted to encode a single transmembrane domain [9] at residues 30–50 but no evidence of an N-terminal signal peptide was detected. BLAST analysis of the protein coding sequence of TEMP determined that orthologous protein sequences were only present in mammalian species. The multiple sequence alignment of these orthologous proteins (Figure 1) revealed that the transmembrane domain is highly conserved across all species with >80% sequence identity. The second highly conserved region near the carboxyl-terminus of the protein encodes a 15 amino acid sequence, EDDDGFIEDNYIQPG, that demonstrates 100% identity across all species. Interestingly, this sequence is also present in the Leucine-rich repeat containing Protein 19 (Lrrc19), a Type I transmembrane protein related to Toll-like receptors involved in the innate immune system [10]. This sequence is highly conserved within Lrrc19 othologs and is located within its cytoplasmic domain. The protein motif observed in both TEMP and Lrrc19 has not previously been characterised, however, the high degree of conservation of this sequence suggests functional importance. Other predicted motifs identified within TEMP and conserved across species included a putative glycosylation site within the amino-terminus and a putative endosome sorting [D/E]xxx[L/I] motif [11] at the carboxy-terminus [4,5,6].

The tissue-specific expression pattern of TEMP in mouse was examined using BioGPS/ SymAtlas [12,13]. TEMP demonstrates a restricted, tissue-specific expression profile to the stomach, kidney, large and small intestines and kidney at levels ten times higher than the median value of the transcript for all of the tissues examined.

Mammalian expression plasmids with a myc epitope located at the amino-terminus of TEMP were transiently expressed in HeLa cells and the whole cell lysate was analysed using Western immunoblotting. TEMP has a predicted molecular mass of 11.5 kDa, increasing to 12.8 kDa with the myc-epitope, however the observed molecular mass of the expressed construct is 24 kDa (Figure 2). The observation of this larger molecular mass could be attributed to post-translational modification most likely N-glycosylation of the conserved site at the amino-terminus. To determine the topology of TEMP with respect to the membrane, the amino-terminal myc-tagged expression construct was transiently transfected into HeLa cells and the topology of the amino-terminus was then investigated in both permeabilised and unpermeabilised cells (Figure 3). An antibody against the sorting nexin 1 (SNX1) protein, a peripheral membrane protein that resides in the cytosol and associates with endosomal membranes [14], was used as an internal control to determine the integrity of the plasma membrane in individual cells. Detection of the myc epitope at the cell surface in unpermeabilised cells indicates the amino-terminus is exposed to the extracellular surface (Figure 3D). The plasma membrane of these surface labelled cells is uncompromised as is demonstrated by an absence of SNX1 labeling (Figure 3E) when compared to the cells permeabilised with 0.1% Triton X-100 where SNX1 labelling is clearly observed (Figure 3B). Collectively, these results indicate that the N-terminus of TEMP is expressed extracellularly. Combining this data with the computational prediction of a single transmembrane domain supports that TEMP has a Type III topology with respect to the plasma membrane [7]. This orientation is consistent with the amino terminus of the protein being exposed to the lumen of intracellular organelles and hence the conserved N-glycosylation motif would be available for post-translational modification. In addition, the motif shared with Lrrc19 would likewise be present in the cytoplasm along with the proposed carboxy-terminal endosome sorting motif.

TEMP was previously demonstrated to localise to the plasma membrane and to intracellular punctate structures using a linear, amino-terminal, myc-tagged expression construct [1]. To determine the nature of the intracellular compartments to which TEMP localises, further co-localisation studies were initially performed with two endosome proteins SNX1 and YFP tagged Rabankyrin-5. SNX1 is a peripheral membrane protein that regulates the correct sorting of receptors at the early endosome [14,15,16]. TEMP clearly co-localises with SNX1 positive structures (Figure 4B–D) demonstrating that it is present on early endosomes. Interestingly, cells expressing TEMP present a clear redistribution of SNX1 labelling with a larger concentration of puncta in the cell periphery (Figure 4C) when compared to untransfected cells (Figure 4A). Rabankyrin-5 is a Rab5 effector that is involved in the fusion of early endosomes [17]. TEMP also clearly co-localises with YFP-Rabankyrin-5, particularly in the cell periphery as is shown in Figure 4F–H. Again, similar to SNX1, the distribution of YFP-Rabankyrin-5 (Figure 4E) is altered upon co-expression with TEMP resulting in its redistribution from a diffuse punctate staining across the entire cell to be concentrated in the cell periphery (Figure 4G). This data demonstrates that TEMP clearly localises to a number of endocytic compartments with the primary evidence demonstrating TEMP is localised to early endosomes. Additionally, expression of TEMP appears to affect the distribution of the early endosomal markers presented.

Although TEMP demonstrates partial colocalisation to early endosomes, a large proportion of punctate structures fail to colocalise. To further investigate this observation colocalisation studies were performed with the Rab proteins to elucidate the precise endosomal sub-compartments to which TEMP localises. Rab proteins are GTPases that regulate membrane traffic and as such are localised to specific subcompartments (reviewed in [18,19,20]). Rab5 is required for transport from the plasma membrane to the early endosome [21,22] and represents a marker for early endosomes. TEMP was co-expressed with GFP-Rab5 (Figure 5A–F). TEMP demonstrates colocalisation with Rab5 in punctate vesicular structures at the periphery of the cell (Figure 5D–F). Overexpression of Rab5 alone can result in the dilation of endosome compartments due to the change in kinetics of subsets of membrane transport pathways [23]. Rab7 represents a well established marker for late endosomes [24]. TEMP was co-expressed with GFP-Rab7 (Figure 5G–L), however, only a few instances of co-localisation to the same structure were observed. Puncta that do demonstrate colocalisation are at the cell periphery (Figure 5J–L), however, the majority of Rab7 is observed in a perinuclear region and typically do not contain TEMP. The colocalisation of TEMP relative to Rab5 and Rab7 is consistent with it associating with early endosome with limited association with late endosomal compartments. To further refine the types of early endosomes TEMP associates with colocalisation studies with Rab4 and Rab11 were performed. Both of these Rabs have been implicated in recycling of the transferrin receptor to the plasma membrane and are markers of the recycling endosome [25,26,27,28,29]. Rab11 has been localised to perinuclear recycling endosomes and in trafficking from the plasma membrane to the Golgi apparatus [29]. TEMP clearly demonstrates colocalisation when co-expressed with YFP-Rab11 as is seen in Figure 5M-R. This colocalisation pattern is particularly apparent in the clusters of endosomes within the cell periphery (Figure 5P–R). Rab4 has been localised to early and recycling endosomes and has been implicated in the sorting of proteins at this stage of endocytosis [25,26,28]. TEMP clearly demonstrates colocalisation when co-expressed with GFP-Rab4 as is observed in Figure 5S–X. Colocalisation is strikingly apparent in enlarged endosomes at the cell periphery (Figure 5V–X). Collectively this subcellular localisation indicates that TEMP associates with both early and recycling endosomes.

We identified that TEMP clearly localises to early and recycling endosomes in fixed cells. To investigate it within live cells we generated a carboxyl-terminal GFP-tagged expression construct. Time-lapse videomicroscopy was performed to determine the dynamic nature of TEMP’s subcellular localisation in transiently transfected HeLa cells. As in fixed cells, TEMP is present on early-/recycling-endosomes concentrated at the cells periphery. In addition, in living cells TEMP was observed on numerous smaller vesicular structures that are observed moving throughout the cell (Figure 6). TEMP-GFP was also clearly localised to tubular endosome structures. It is evident from the time-lapse videomicroscopy that some of these tubular structures are dynamic whereas others appear to be more static in their nature. The number and nature of the tubules appears to vary from cell to cell. More dynamic tubules are observed trafficking from the perinuclear region to the cell periphery and vice versa. To determine if the microtubule network is responsible for the peripheral nature of these clusters of endosomes, HeLa cells were transiently transfected with TEMP and expressed for 16 h prior to 10 μM nocodazole treatment, subsequent fixation and immunodetection of the microtubule network (Figure 7). The disruption of the microtubule network altered the subcellular localisation of TEMP with loss of the concentration of peripheral endosomes, resulting in their even redistribution throughout the cytoplasm (Figure 7G/J).

General sequence characteristics including length, predicted InterPro domains and predicted transmembrane domains were collated from the LOCATE database [5,6]. Independent predictions were obtained using TMHMMv2.0 [30] for transmembrane domains, SignalP3.0 [31] for signal peptides and InterProScan [32,33,34] for the prediction of protein domains. Further details including putative glycosylation sites were obtained from the University of California Santa Cruz (UCSC) proteome browser [35,36]. SymAtlas developed by the Genomics Institute of the Novartis Research Foundation (GNF) [12] was used to analyse the gene expression patterns of the proteins investigated. Orthologs were analysed by submitting the query sequence to the BLAST tool [37,38]. The sequences were analysed against the non-redundant dataset provided by NCBI and corresponding orthologs were identified. These orthologs were subsequently aligned in a multiple sequence alignment using the AlignX tool in VectorNTI^® (Invitrogen, USA) using the default parameters.

Expression constructs comprised of an amino-terminal cytomegalovirus (CMV) promoter, an amino-terminal, myc-tagged protein coding sequence (CDS) and two C-terminal Simian Virus 40 (SV40) poly-adenylation sequences were generated using the Megaprimer PCR protocol outlined in [1]. The subsequent PCR product was subsequently, T-A cloned into pGEM^®-T (ProMega, USA) according to the manufacturer’s instructions.

RIKEN clone 2610528J11 (PA76377.1) was used as a Template to amplify the open reading frame using primers 5'-CCC ATC CCG AAT TCG CCA CCA TGA TTG GAG GAA ACA C-3' and 5'-CCC ATC CCG GAT CCG GGA GAG AGA AGT GGT CCC G-3' which incorporate a 5' EcoRI site and a 3' BamHI site to the PCR product. The purified PCR product and pEGFP-N1 (Clontech Laboratories Inc., USA) were digested with EcoRI (New England Biolabs, USA) and BamHI (New England Biolabs, USA) and the subsequent products were ligated together using T4 DNA ligase (Invitrogen, USA).

GFP-Rab5 [25], YFP-Rab11 [25], GFP-Rab7 [39], YFP-Rabankyrin-5 [17] and GFP-Rab4 (kindly donated by Dr Frederic Meunier, The University of Queensland).

Mouse monoclonal antibodies were used to detect the myc-epitope tag (Cell Signalling Technology, USA). Alternatively, a rabbit polyclonal antibody was used to detect the myc-epitope tag (Cell Signalling Technology, USA). Monoclonal mouse antibodies used included anti-α-tubulin (clone DM1A, mouse ascites fluid) (Sigma-Aldrich, USA), anti-Sorting Nexin 1 (SNX1) (BD Transduction Laboratories, USA). The following secondary antibodies were used for immunofluorescent labelling: goat anti-rabbit Alexa Fluor 488 (Molecular Probes, USA), goat anti-mouse Cy3 (Molecular Probes), goat anti-mouse Alexa Fluor 488 (Molecular Probes, USA) and goat anti-mouse Alexa Fluor 647 (Molecular Probes, USA).

HeLa cells and MDCK cells were cultured in Minimum Essential Medium (Eagle) (Life Technologies Inc., USA) supplemented with 2 mM L-glutamine and Earle’s BSS adjusted to contain final concentrations of 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate and 10% fetal bovine serum in 5% CO₂ and 95% air at 37 °C. Subconfluent cell monolayers were transiently transfected with expression constructs using Lipofectamine 2000 (Life Technologies Inc., USA) and OptiMem (Life Technologies Inc., USA) as per the manufacturer’s instructions.

Proteins were run on 10% Sodium Dodecyl Sulfate Poly-Acrylamide Gels (SDS-PAGE) gels prior to transfer onto PVDF membranes (Millipore, USA) according to the manufacturer’s instructions. Immunoblotting was subsequently performed as described in [40]. Anti-myc antibodies were used to detect the epitopes in conjunction with sheep anti-mouse Ig horseradish peroxidase conjugated secondary antibodies (Silenus, Australia).

Coverslips of sub-confluent HeLa cells were rinsed with ice-cold CO₂ independent media (Life Technologies Inc.) and then placed on a drop of ice-cold CO₂ independent media supplemented with 0.1% BSA on ice for 5 min. Cells were then placed on 30 μL of primary antibody diluted in ice-cold CO₂ independent media/0.1% BSA for 30 min at 4 °C prior to fixation with 4% paraformaldehyde for 20 min at room Temperature. Cells were then washed three times with PBS prior to quenching with 50mM ammonium chloride in PBS for 15 min at room Temperature. Cells were then incubated in blocking solution composed of 0.2% BSA and 0.2% fish skin gelatin in PBS for 10 min at room Temperature prior to further incubation with 25 μL of the secondary antibody in blocking solution for 30 min at room Temperature. Cells were then washed thrice in PBS prior to mounting with MO-WIOL.

Indirect immunofluorescence was performed as described [1]. Cell monolayers were treated with 10 μM nocodazole (Sigma-Aldrich, USA) for 30 min prior to fixation.

Representative images of the observed localisation patterns for all fixed cell experiments were performed on a Zeiss Axiovert 200 M SP LSM 510 META (Zeiss, Germany) inverted, laser scanning confocal microscope with appropriate band pass filter settings. Data was analysed using the LSM 510 META (Zeiss, Germany) software and images were prepared using Adobe Photoshop 7.0 (Adobe Systems, USA).

Cells were prepared in MatTek glass bottom tissue culture dishes (MatTek Corporation, USA) and grown under conditions as previously described. Prior to analysis with time-lapse videomicroscopy, cells were washed with PBS, and placed into CO2-independent media (Invitrogen, USA) supplemented with 5% fetal calf serum and 2 mM L-glutamine (Life Technologies Inc., USA). Videomicroscopy was then performed using an Olympus IX-81 OBS epifluorescence microscope attached to a heated chamber (Solent Scientific, UK) at 37 °C. Images were captured with the 60X objective at varying exposure times and intervals using Cell^R software (Olympus, Japan). ImageJ software [41] was used to process image stacks.