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Human Molecular Genetics Advance Access originally published online on March 24, 2005
Human Molecular Genetics 2005 14(10):1261-1270; doi:10.1093/hmg/ddi137
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Comparative evolutionary analysis of VPS33 homologues: genetic and functional insights

Paul Gissen1,2, Colin A. Johnson1, Dean Gentle1, Laurence D. Hurst3, Aidan J. Doherty4, Cahir J. O'Kane5, Deirdre A. Kelly2 and Eamonn R. Maher1,*

1Section of Medical and Molecular Genetics, University of Birmingham, Birmingham B15 2TG, 2The Liver Unit, Birmingham Children's Hospital, Birmingham B4 6NH, UK, 3Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK, 4UK Genome Damage and Stability Centre, University of Sussex, East Sussex BN1 9RQ, UK and 5Department of Genetics, University of Cambridge, Downing Street CB2 3EH, Cambridge, UK

* To whom correspondence should be addressed. Tel: +44 1216274434; Fax: +44 1214141695; Email: e.r.maher{at}bham.ac.uk

Received December 21, 2004; Accepted March 21, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
VPS33B protein is a homologue of the yeast class C vacuolar protein sorting protein Vps33p that is involved in the biogenesis and function of vacuoles. Vps33p homologues contain a Sec1 domain and belong to the family of Sec1/Munc18 (SM) proteins that regulate fusion of membrane-bound organelles and interact with other vps proteins and also SNARE proteins that execute membrane fusion in all cells. We demonstrated recently that mutations in VPS33B cause ARC syndrome (MIM 208085 [OMIM] ), a lethal multisystem disease. In contrast, mutations in other Vps33p homologues result in different phenotypes, e.g. a mutation in Drosophila melanogaster car gene causes the carnation eye colour mutant and inactivation of mouse Vps33a causes buff hypopigmentation phenotype. In mammals two Vps33p homologues (e.g. VPS33A and VPS33B in humans) have been identified. As comparative genome analysis can provide novel insights into gene evolution and function, we performed nucleotide and protein sequence comparisons of Vps33 homologues in different species to define their inter-relationships and evolution. In silico analysis (a) identified two homologues of yeast Vps33p in the worm, fly, zebrafish, rodent and human genomes, (b) suggested that Carnation is an orthologue of VPS33A rather than VPS33B and (c) identified conserved candidate functional domains within VPS33B. We have shown previously that wild-type VPS33B induced perinuclear clustering of late endosomes and lysosomes in human renal cells. Consistent with the predictions of comparative analysis: (a) VPS33B induced significantly more clustering than VPS33A in a renal cell line, (b) a putative fly VPS33B homologue but not Carnation protein also induced clustering and (c) the ability to induce clustering in renal cells was linked to two evolutionary conserved domains within VPS33B. One domain was present in VPS33B but not VPS33A homologues and the other was one of three regions predicted to form a t-SNARE binding site in VPS33B. In contrast, VPS33A induced significantly more clustering of melanosomes in melanoma cells than VPS33B. These investigations are consistent with the hypothesis that there are two functional classes of Vps33p homologues in all multicellular organisms and that the two classes reflect the evolution of organelle/tissue-specific functions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The functional analysis of novel genes represents a major challenge to human genetics research. Recently, we demonstrated that a rare autosomal recessive disorder, ARC syndrome, characterized by neurogenic arthrogryposis multiplex congenita, renal tubular dysfunction and neonatal cholestasis with bile duct hypoplasia and low gamma glutamyl transpeptidase (gGT) activity was caused by mutations in the human VPS33B gene (1Go). Although the function of the VPS33B protein has not been studied previously in humans, the yeast homologue, Vps33p, a class C vacuolar protein sorting protein, is required for vacuolar biogenesis and has a key role in protein trafficking from Golgi to vacuole (2Go). Furthermore, yeast mutants of the class C vps proteins (consisting of Vps11p, Vps16p, Vps18p and Vps33p) display severe intracellular acid–base imbalance, amino acid pool deficiency and temperature-sensitive growth failure (3Go). These findings suggested that the ARC phenotype might be explained by abnormal organelle biogenesis. Consistent with this hypothesis, we demonstrated that (a) ectopically expressed VPS33B localized to LAMP1-positive late endosomes and lysosomes and (b) overexpression of VPS33B in human renal cells caused perinuclear clustering of late endosomes and lysosomes (1Go). Late endosomes or multivesicular bodies are membrane bound organelles which share their biogenesis pathway with lysosomes and like melanosomes belong to the class of lysosome-related organelles (LRO) (4Go,5Go). Late endosomes constitute the penultimate stage in the endocytic transport of proteins to lysosomes and are also involved in recycling of membrane proteins. Perinuclear clustering of late endosomes has been shown to occur in the presence of over-expressed class C vps protein homologues and their interacting partners (6Go).

Inactivation of yeast Vps33 homologues in other species is associated with phenotypes significantly different from ARC. Thus in Drosophila, a hypomorphic allele of the Vps33 homologue carnation (car) causes the carnation eye colour phenotype. The car gene product (Carnation) localizes to endosomal compartments and is a homolog of SM regulators of membrane fusion (7Go). In mice, a mutation in the mVps33a gene caused the buff (bf) mouse phenotype that is characterized by hypopigmentation and a mild platelet storage pool deficiency and has been proposed as an animal model for Hermansky–Pudlak syndrome (8Go). Apart from the platelet dysfunction, the bf mouse does not have any other phenotypic features in common with ARC syndrome patients. VPS33A and VPS33B share 31% identity and 51% similarity, and are much more closely related to each other than to the yeast Vps33p (24% identity and 41% similarity for VPS33B, and 27% identity and 46% similarity for VPS33A). However, as both genes are ubiquitously expressed, it would appear that VPS33A and VPS33B have evolved different functions.

In yeast, the class C vps proteins, together with Vps39p and Vps41p form a homotypic vacuolar protein sorting (HOPS) complex which has a role in the recruitment of the membrane-bound organelles for the fusion events required for endo/exocytosis and secretion (9Go,10Go). Recent findings suggest that the HOPS complex and the SM-like family of proteins are essential for vesicular trafficking and may be crucial for ensuring the specificity of SNARE-mediated membrane fusion (11Go–15Go). SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptor or SNAP receptor proteins) are membrane bound proteins present on both vesicular (v-SNARE) and target (t-SNARE) membranes. The v- and t-SNAREs form a complex that pulls opposing membranes together (16Go,17Go). SNARE function is mediated by upstream regulators and tethering factors such as the HOPS complex, which also interacts with members of the family of Rab GTPases and SM proteins. These tethering factors bring the two vesicles into close proximity thus determining the specificity of the interaction.

To gain insights into the evolution of Vps33p homologues in multicellular organisms, we undertook nucleotide and protein sequence comparisons in a variety of species and assembled a phylogenetic tree. This analysis suggested that VPS33A and VPS33B diverged at an early stage of metazoan evolution and that Carnation is more closely related to VPS33A than VPS33B. This finding prompted us to search for a Drosophila orthologue of human VPS33B. We investigated the function of the VPS33B candidate orthologue by determining its ability to induce perinuclear clustering of late endosomes and lysosomes in human cells. We also investigated the functional importance of potential protein binding motifs in VPS33B predicted by comparative sequence analysis and compared the effects of VPS33A and VPS33B overexpression on clustering of melanosomes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Homology searching and phylogenetic analysis
Basic Local Alignment Search Tool (BLAST) homology search using human VPS33B as a query identified the following known, annotated proteins: Saccharomyces cerevisiae Vps33p (NP-013500), Drosophila melanogaster Carnation protein (NP-523410), Homo sapiens VPS33A and VPS33B (NP-061138 and NP-075067), Mus musculus mVps33A and mVps33b (NP-084205 and NP-835171) and Rattus norvegicus r-Vps33a and r-Vps33b (NP-075250 and NP-071622) proteins. In addition, BLAST searches identified two unannotated homologues from Danio rerio (ENSDARP00000013152 and ENSDARP00000006820), a novel fly Vps33p homologue CG5127-PA (NP-651395) and two Caenorhabditis elegans homologues known as C56C10.1 (NP-495342) and SLP-1 (P34260 [GenBank] ). Protein sequence alignments demonstrated that among the mammalian VPS33B and VPS33A homologues, VPS33B orthologues were more conserved than VPS33A orthologues. Thus human VPS33B has 96% pairwise identity and 97% similarity (and no gaps) with rat r-Vps33b, but VPS33A has 78% identity and 82% similarity (and 4% gaps) to rat r-Vps33a orthologues (Fig. 1A and B). To study the evolutionary inter-relationships of VPS33A and VPS33B homologues further, we performed both a Bayesian (Fig. 1C) and a quarter puzzling maximum likelihood (Fig. 1D) estimation of phylogeny. Thus we created two phylogenetic trees rooted with a Vps33p from multiple protein sequence alignment (Fig. 1C and D). In both cases, the tree divided into two branches containing orthologues of VPS33A and VPS33B in all multicellular organisms. This suggests that either (a) a duplication of the gene occurred because the divergence of animals from fungi relatively close to the base of the animal lineages or (b) the duplication was ancestral to both fungi and animals but lost in the lineage leading to yeast. Carnation was predicted under both methods to be the orthologue of VPS33A, whereas Drosophila CG5127 is orthologous to VPS33B.






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  		Figure 1. Alignment of (A) VPS33A and (B) VPS33B orthologues in S. cerevisiae, C. elegans, D. melanogaster, D. rerio, M. musculus, R. norvegicus and H. sapiens. Phylogenetic trees for the homologues of the yeast Vps33p protein (C) using Bayesian analysis (50 changes represent an absolute number of changes) and (D) using quartet puzzling maximum likelihood analysis. The horizontal bar represents a 0.1 evolutionary distance (slightly <10% divergence to allow for multihit correction).

 
Evolutionary conservation of VPS33A and VPS33B
We used overlapping sliding window analysis with a block-size of 90 nucleotides, with jumps of 30 nucleotides, and for each window calculated the non-synonymous/synonymous substitution ratio (Ka/Ks) among human, mouse and rat VPS33B orthologues. This analysis detected strong stabilizing selection across the whole of the gene, with particularly stable regions in the N-terminal 60 codons (nucleotides ~0–180) and between codons 250–500 (nucleotides ~750–1500) (Fig. 2). Similarly high conservation was found between human and mouse VPS33A orthologues (data not shown). Significant sequence differences between codons 100 and 250 were present between the human and rat VPS33A orthologues, and this region was also the least conserved in VPS33B. Sequence alignment of VPS33B and VPS33A proteins revealed an overall pairwise protein similarity and identity of 51 and 31%, respectively (48 and 27%, respectively, in the N-terminal 15–60 codons and 52 and 28%, respectively, between codons 240–450) and an insertion of 31 amino acids (450–480) in VPS33B that was absent from VPS33A (Fig. 3A).



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  		Figure 2. Comparison of non-synonymous/synonymous substitution ratio (Ka/Ks) of human VPS33B versus R. norvegicus (red line) and M. musculus (blue line) orthologues. An overlapping sliding window of 90 nucleotides was used with jumps of 30 nucleotides. A, B and C are predicted t-SNARE binding motifs. D is an insertion of 29 codons not present in VPS33A. Mutations that are predicted to be pathogenic in ARC syndrome are indicated as follows: missense substitution (M), frameshift (F), splice-junction (S) and nonsense (N). Ka/Ks values are shown on the y-axis, and the x-axis displays the window midpoint in nucleotides starting from the first nucleotide of the starting codon.

 


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  		Figure 3. The predicted structure of the VPS33B protein complexed with the t-SNARE syntaxin1. VPS33B is shown in blue and syntaxin1 in green. (A) The region deleted in the construct VPS33B1-438 is shown in red and the 29 amino acid insertion absent in VPS33A in orange. (B) The t-SNARE binding motifs are shown in pink and the L30P mutation site in red. (C) Close-up of the L30P mutation which is predicted to disrupt alpha helix 1 (arrow) and hence the N-terminal binding site for the t-SNARE.

 
VPS33B structure–function predictions
Using the previously reported crystal structure of the interaction between neuronal-Sec1 and Syntaxin1 proteins in rat (18Go) as a model, we searched VPS33B for putative binding sites for a Syntaxin1 homologue. This revealed three putative binding motifs (A, B and C) predicted to interact with a Syntaxin1 homologue (Fig. 3B). These predictions were consistent with the results of the Ka/Ks ratio analysis as each of the predicted binding motifs were contained within regions under strong stabilizing selection (A between codons 35 and 60, B between codons 260 and 275 and C between codons 310 and 340). In addition, although most VPS33B mutations in ARC syndrome are predicted to be null mutations, we have previously identified a missense substitution at L30P in a patient with severe ARC syndrome. Structural modelling of this mutation predicted that the substitution would distort the putative N-terminal binding site A, consistent with the evolutionary conservation analysis (Fig. 3C).

Function of human Vps33p homologues and vesicular clustering
In order to corroborate the results of the evolutionary conservation analysis and structure predictions, we investigated the ability of the putative Drosophila VPS33B orthologue CG5127, Carnation and human VPS33A, to induce clustering of vesicles in a renal cell carcinoma (RCC4) cell line. There was clear evidence of vesicular clustering (65%, 26 of 40 cells) when compared with mock-transfected cells (0%, zero of 40 cells), 48 h after transfection of RCC4 cells with wild-type VPS33B (WT). Transfection of RCC4 cells with wild type VPS33A induced clustering in a smaller number of cells (12.5%, five of 40 cells) (P<10–6 versus VPS33B). The novel putative fly VPS33B orthologue (CG5127) induced clustering in 25% of the cells (10 of 40 cells) whereas empty vector control and Carnation had no effect (each 0/40 cells, P=0.02 for CG5127 versus carnation, and for CG5127 versus empty vector) (Fig. 4). No clustering was seen in cells transfected with a truncated VPS33B(1–438), and reduced clustering effect (12.5%, P<10–6 versus WT) was seen in the cells transfected with a VPS33B({Delta}46–52) with a deleted predicted syntaxin-binding region A. We also investigated the effect of the deletion of 31 amino acids present in VPS33B and absent in VPS33A. Transfection of the cells with pCMVHA+VPS33B({Delta}450–480) had a significantly reduced clustering effect (10%, P<0.001 versus WT).



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  		Figure 4. Clustering of LAMP1-positive organelles. RCC4 cells were transfected with a pCMVHA+Carnation (A–C) or pCMVHA+CG5127 (D–F) constructs showing LAMP1 staining (A and D), HA staining (B and E) and merge (C and F, LAMP1 red, HA green and DAPI blue). Scale bar 5 µm. The arrow in D indicates a cell with clustered LAMP1-positive organelles. (G) A bar chart, showing the percentage of transfected RCC4 cells displaying clustering. *P=0.02 for CG5127 versus both empty vector and Carnation. **P<10–6 versus VPS33B.

 
To investigate whether the clustering effect is organelle/tissue-specific, we overexpressed VPS33B and VPS33A in mouse malignant melanoma cells (Fig. 5). Overexpression of VPS33A caused clustering of melanosomes in 34% (86 out of 250 cells, P<10–13 versus empty vector where clustering was observed in 8%, 20 of 250 transfected cells). Overexpression of VPS33B caused clustering of melanosomes in 15% of cells (37 of 250 cells, P<0.05 versus empty vector and P<10–7 versus VPS33A).



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  		Figure 5. Clustering of melanosomes in mouse malignant melanoma cells transfected with pCMVHA+VPS33A construct. (A) Blue DAPI staining of nuclei, black melanosomes on phase-contrast microscopy. (B) The same image showing co-localization of the construct stained with anti-HA antibody (red) and the melanosome cluster (black). Arrows point to the cluster of melanosomes.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We identified two homologues of the yeast Vps33p protein in all multicellular organisms studied. Each of these homologues contains a Sec1-like domain and corresponds to one of the two distinct classes of the SM-like protein family, which is highly conserved among species. Yeast, worm, zebrafish and mammalian genomes contain between four and seven SM genes, whose products display significant homology along the whole sequence. Although it has been suggested that overexpression of a homologue from the same species cannot substitute for a null mutation in another SM protein coding gene, it is clear that an orthologue from another species can partially restore its function (19Go–22Go). For example, mouse munc18-1 rescues the unc-18 null mutant phenotype in C. elegans, but munc18-2 cannot. Most of the loss-of-function mutations described in SM proteins lead to lethal phenotypes, underlining the importance of these proteins in vesicular trafficking (11Go). The function of the SM proteins is not clear but they are known to associate with target SNAREs (principally, syntaxin proteins) and also tethering factors such as the HOPS complex proteins (23Go–25Go). Recent findings suggest that despite high sequence homology between SM proteins they form complexes with specific SNAREs and play crucial role in determining the specificity of the SNARE-complex assembly (18Go,26Go). In yeast, Vps33p has been shown to interact directly with vacuolar SNARE Vam3p and its homologue late endosomal SNARE Pep12p (2Go,27Go). The latter also interacts with another SM protein Vps45, which is exclusively involved in the traffic of biosynthetic vesicles from Golgi. Although this finding is surprising, this dual SM action on Pep12p may be explained if the presence of Vps33p is a distinguishing feature of the endocytic vesicles originating in the cell membrane. This suggestion is confirmed by the fact that the two SM proteins cannot be used interchangeably (27Go). In parallel, functional and genetic studies in yeast implicated HOPS complex in protein trafficking along the recycling pathway to and from late endosomes (28Go). Furthermore, Vps33p mutants displayed abnormal recycling of the plasma membrane proteins. These findings may explain the nature of the molecular pathophysiology of patients with ARC syndrome. Thus mutations in human VPS33B gene are associated with abnormal localization of plasma proteins in polarized cells. This may occur due to loss of interaction with a SNARE protein at the late endosomal stage and may lead to their accumulation in the cytoplasm or mislocalization on the plasma membrane. This hypothesis requires further investigation.

The presence of two VPS33 homologues suggests that they have distinct roles in normal cellular functions. The phenotype of the bf mouse suggests abnormal biogenesis and transport of the melanosomes and platelet granules. In contrast, there is no evidence of albinism or abnormal organellar biogenesis in patients with ARC syndrome. However, ARC syndrome patients develop hepatocyte accumulation of lipofuscin granules and abnormal localization of the membrane proteins in polarized epithelial cells of the liver and kidneys (1Go). Late endosomes, melanosomes, lipofuscin and platelet storage granules, all belong to a class of the LRO that share their biogenesis pathways. Thus it appears that VPS33B and VPS33A orthologues function in the LRO-specific pathways determined either by a tissue-specific or other, yet unknown factors.

We identified a putative novel homologue of yeast Vps33p (CG5127) in Drosophila and predicted that it is functionally closer to the VPS33B. To confirm this we overexpressed Carnation and CG5127 in the human RCC4 cell line. We compared the percentage of cells with the distinct morphology of clustered LAMP1-positive organelles with that achieved by overexpression of VPS33B and VPS33A. These experiments identified that in RCC4 cells VPS33A has a much lower capacity to produce clustering than VPS33B (12.5 versus 65%, P<<0.05). CG5127 retained the ability to induce clustering, unlike Carnation protein (25 versus 0%). Thus these findings are entirely consistent with the in silico prediction that CG5127 is a VPS33B orthologue.

In contrast, analysis of clustering of melanosomes in F1P43 mouse melanoma cell line found that VPS33A had significantly more effect than VPS33B (34 versus 15%, P<10–7). This observation demonstrates that the ability of a Vps33p homologue to induce clustering is organelle and/or tissue-specific. Our results emphasize the conservation and specificity of the pathway for each Vps33 homologue. It is interesting to note that there is more evolutionary conservation between human and rodent VPS33B orthologues than the VPS33A orthologues, and that the phenotypic consequences of VPS33B inactivation are more severe than for VPS33A.

Ka/Ks analysis of VPS33B identified five regions of strong stabilizing evolutionary influence. To gain insights into the possible relevance of these to VPS33B function, we undertook structural predictions based on the crystallographic study of the nSec1–Syntaxin1 complex (18Go). Three predicted binding sites for syntaxin corresponded to three of the five regions with strong stabilizing selection identified by the Ka/Ks analysis (labelled A, B and C). Furthermore, the one known pathogenic VPS33B missense mutation (L30P, which causes severe ARC phenotype) is predicted to disrupt the putative N-terminal binding site A. The other two evolutionary stable regions were not predicted to be implicated in syntaxin binding. However, region D corresponded to a 31 amino acid insertion present in VPS33B and not VPS33A, suggesting a key role in VPS33B function. Accordingly both VPS33A and a VPS33B protein-lacking region D demonstrated impaired or absent ability to induce vesicular clustering when compared with wild-type VPS33B.

Clustering and fusion of LAMP1-positive organelles such as late endosomes, lysosomes or pigment granules in mammalian cells may be induced by a variety of mechanisms. Thus overexpression of Rab7, Rab7-interacting lysosomal protein, Vps18, Vps39 and Vps33b, and a dominant-negative mutant of Rab27a can all induce clustering. The ability to induce clustering has been used to implicate candidate proteins in vesicular trafficking processes (6Go,29Go–32Go). Although the precise mechanisms by which these interventions induce clustering have not been defined they are known to involve cell motors and components of the actin cytoskeleton such as actin, ezrin and specific unconventional myosins, all of which surround clusters (33Go). Although we have demonstrated previously that VPS33B could induce clustering, the function of VPS33A has not been investigated before. To our knowledge this is the first demonstration of the organelle/tissue-specific nature of clustering. Here we suggest that the interaction between VPS33B and syntaxin may be important in clustering, as deletion of the predicted N-terminal syntaxin binding site diminished the ability to cause clustering by overexpression. However, deletion of the C-terminal portion of the gene also impaired VPS33B-induced clustering. This region of VPS33B is not predicted to be involved in syntaxin binding but does contain the stretch of 31 amino acids that is absent in VPS33A. Thus, the presence of this motif correlated with the ability to induce clustering and may relate to interactions with other proteins such as Rab 7 or VPS18. Further studies to define the functional significance of the VPS33B C-terminal motif will provide insights into the organelle/tissue-specific effects of VPS33A and VPS33B and their differing roles in metazoans.

Our findings demonstrate how a combination of in silico evolutionary analysis and functional studies can identify candidate regions critical for normal protein function. Recent discoveries of the role of vesicular trafficking genes in human disease emphasize the importance of these pathways for cell homeostasis and the importance of research in model organisms to human genetics.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Homology search
A homology search was performed using standard BLAST algorithms on the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/) against the non-redundant database, FlyBase (http://flybase.bio.indiana.edu/), Wormbase http://www.wormbase.org/), the Zebrafish genome resource on the NCBI server (http://www.ncbi.nlm.nih.gov/genome/seq/DrBlast.html), Saccharomyces Genome Database (http://www.yeastgenome.org/), UCSC genome browser http://genome.ucsc.edu/cgibin/hgGateway) and Ensembl genome browser (http://www.ensembl.org/).

Multiple sequence alignment
Multiple sequence alignment for the Vps33p protein homologues was performed using ClustalW and Boxshade software on the Baylor College of Medicine website (http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html). For the phylogenetic analysis the alignment was further purged using Gblocks to extract regions of high quality informative alignment (34Go). Default settings were employed.

Phylogenetic tree generation
A phylogenetic tree was created using MrBayes software downloaded from http://morphbank.ebc.uu.se/mrbayes/. MrBayes is a program for the Bayesian estimation of phylogeny, which is based upon the posterior probability distribution of the trees, conditioned on the observations (35Go). The posterior probability distribution of trees is calculated using a simulation technique, Markov chain Monte Carlo (MCMC). The analysis was performed on the protein alignment using a mixed model for amino acid evolution. The analysis was run for a million generations, resulting in 10 000 trees. A conservative burn in of 7000 trees was employed. The 50% consensus rule tree was then constructed on the remaining 3000 trees with the support values given being the proportion of the trees supporting the consensus tree. A second tree was created from the same alignment using Tree Puzzle, a quartet puzzling maximum likelihood method (36Go). The Mueller Vingron model of amino acid evolution was employed with one fixed and eight gamma rates. The nodal values are the support values estimated by the method.

Ka/Ks sliding window analysis
Analysis of the molecular evolution of the VPS33A and VPS33B genes was undertaken using a sliding window approach for the human, mouse and rat genes. The variables measured in each pairwise comparison were: (a) Ka, the rate of non-synonymous substitution per non-synonymous site (a substitution being a mutation that has gone to fixation) and (b) Ks, the rate of synonymous substitution per synonymous site. The ratio Ka/Ks provides a measure of the form of selection occurring. The meaning of particular values of Ka/Ks ratios is: Ka/Ks=1, sequence is evolving neutrally; Ka/Ks<1, the sequence is under stabilizing selection; Ka/Ks>>1, the sequence is under directional selection. Most analyses result in ratios of between 0.1 and 0.2. We used overlapping sliding window with the size of the block of 90 nucleotides and jumps of 30 nucleotides. The protocol of Li (37Go) was employed to estimate Ka and Ks in each window.

Structural modelling
Structural representation of the VPS33B and VPS33A proteins was performed using RasMol, a program for molecular graphics visualization (www.openrasmol.org).

Molecular cloning of the Vps33p homologues
We obtained human cDNA by RT–PCR amplification of the total RNA from a healthy subject. Forward primers for VPS33B and VPS33A genes included the start codon and the reverse primers contained the native stop codon (all primer sequences available on request). A VPS33B construct with a deleted putative N-terminal binding site (deleted amino acids 46–52 MSPLDRI) was made using BglII restriction endonuclease and PCR of the replacement insert. A VPS33B deletion construct VPS33B({Delta}450–480) was made to remove the amino acids absent in VPS33A by PCR of the flanking DNA with the primers to create a BamHI restriction site. The two flanking fragments were then ligated and inserted into the pCMVHA vector. A VPS33B(1–438) construct was made by PCR including start codon and the stop codon mimicking the truncating mutation R438X. cDNAs were cloned into the pCMVHA expression vector (Invitrogen).

We cloned cg5127 and carnation cDNA by RT–PCR amplification of total RNA from D. melanogaster embryos (a kind gift from Dr Badenhorst, University of Birmingham). To allow subcloning of the cDNA fragments into the pCMVHA expression vector, we designed the forward primers with EcoRI (VPS33B, Car, cg5127) or Sal1 (VPS33A) restriction sites and reverse primers with a Kpn1 restriction sites. All constructs were verified by sequencing using an ABI 3730 DNA Sequencer. Expression of epitope-tagged protein following transfection of the constructs was verified by western blotting with a monoclonal anti-HA antibody (Sigma Aldrich).

Cells and antibodies
We grew human adult RCC4 and mouse malignant melanoma F1P43 cells in Dulbecco's minimal essential medium supplemented with 5% fetal calf serum (Sigma Aldrich). F1P43 cell line was a generous gift from Dr Elena Sviderskaya (St George's Hospital Medical School, University of London, UK). We obtained monoclonal antibodies against LAMP1 (Developmental Studies Hybridoma Bank, University of Iowa). Other antibodies were obtained commercially.

Transient transfection and immunofluorescence
For transient transfection experiments, we grew RCC4 cells and malignant melanoma F1P43 cells on glass coverslips to 80% confluence and transfected them with 500 ng of plasmid DNA. RCC4 cells were transfected with pCMVHA empty vector control, pCMVHA+VPS33B, pCMVHA+VPS33B({Delta}46–52), pCMVHA+VPS33B(1–438), pCMVHA+VPS33B({Delta}450–480), pCMVHA+VPS33A, pCMVHA+Car, pCMVHA+CG5127) constructs using Effectene reagent (Qiagen, GmbH) for 24 h. We then removed transfection complexes and allowed cells to grow for another 24 h in normal medium. We washed RCC4 cells in phosphate buffered saline (PBS), fixed them in 4% paraformaldehyde in PBS for 30 min and permeabilized them in PBST buffer [PBS, 10% (v/v) normal swine serum, 0.1% (v/v) Tween 20]. We detected endogenous LAMP1 using mouse monoclonal antibodies diluted 1:100 in PBST. We detected HA antigen using rabbit polyclonal anti-HA antibody (Sigma Aldrich) at 1:100 in PBST. We detected primary antibodies using TRITC-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG antibodies diluted 1:100 in PBST. All antibodies were applied at room temperature in as moist chamber. Finally we applied antifade (Vectashield, Vector Laboratories) containing DAPI (2 µg/ml). All experiments were performed in triplicate.

F1P43 cells were transfected with pCMVHA empty vector control, pCMVHA+VPS33B and pCMVHA+VPS33A as described previously. We detected HA antigen using monoclonal anti-HA antibody (Sigma Aldrich) at 1:100 in PBST. We detected primary antibodies using TRITC-conjugated anti-mouse IgG.

We visualized the images with a Photometrics SenSys KAF 1400-G2 CCD fitted to a Zeiss Axioplan epifluorescence microscope. Melanosomes were visualized using phase contrast microscopy on the same system using an oil immersion objective. We captured images using SmartCapture 2 software (Digital Scientific) running on a Macintosh G4 computer.

Detection of clustering and statistical analysis
The captured images were analysed using Adobe Photoshop 6.0 software. For the assessment of clustering in RCC4 cells, the image of a single cell was isolated and the total number of pixels and the median level of red luminosity (the intensity of the red signal) were detected. The number of pixels occupied by the red signal with the intensity of above the median level of luminosity was recorded (median.pxls.). Clustering (Q) was measured as a fraction of the cell occupied by the red signal of above median intensity (Q)=median.pxls/total.pxls). We defined clustered cells as those with a Q-value that was less than two standard deviations compared with the average value of Q in control cells. To standardize the level of expression of transfected construct in each individual cell, we only selected cells with the level of green luminosity within one standard deviation from the mean of the positive control (VPS33B wild-type construct). There was no significant difference between expression levels in the selected cells between different transfections.

For the assessment of melanosome clustering in F1P43 cells, we observed the number of cells with pigmented clusters in the field of view. The transfection efficiency was estimated by measuring the red signal and no significant difference was found between VPS33A and VPS33B. The significance of the result was assessed by Fisher's exact test.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Professor J. Paul Luzio for helpful discussion and reading of the manuscript. This work was supported by grants from Children Living with Inherited MetaBolic conditions (CLIMB) charity and Birmingham Children's Hospital Research Foundation. P.G. is a WellChild and RCPCH Research Fellow.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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