Comparison of human solute carriers

Abstract

Solute carriers are eukaryotic membrane proteins that control the uptake and efflux of solutes, including essential cellular compounds, environmental toxins, and therapeutic drugs. Solute carriers can share similar structural features despite weak sequence similarities. Identification of sequence relationships among solute carriers is needed to enhance our ability to model individual carriers and to elucidate the molecular mechanisms of their substrate specificity and transport. Here, we describe a comprehensive comparison of solute carriers. We link the proteins using sensitive profile-profile alignments and two classification approaches, including similarity networks. The clusters are analyzed in view of substrate type, transport mode, organism conservation, and tissue specificity. Solute carrier families with similar substrates generally cluster together, despite exhibiting relatively weak sequence similarities. In contrast, some families cluster together with no apparent reason, revealing unexplored relationships. We demonstrate computationally and experimentally the functional overlap between representative members of these families. Finally, we identify four putative solute carriers in the human genome. The solute carriers include a biomedically important group of membrane proteins that is diverse in sequence and structure. The proposed classification of solute carriers, combined with experiment, reveals new relationships among the individual families and identifies new solute carriers. The classification scheme will inform future attempts directed at modeling the structures of the solute carriers, a prerequisite for describing the substrate specificities of the individual families.

Figure 1

Structure-based classification of solute carriers. A: Structures of solute carriers and their homologs representing the currently known structural classes. The folds include (i) the NSF-like fold, (ii) the ammonium transporter fold, (iii) the MFS general substrate transporter fold, (iv) the mitochondrial carrier fold, and (v) the proton glutamate symport protein fold. B: The relationships between the structures are visualized using cytoscape, based on pairwise structural alignment scores computed by SALIGN. Each link represents a pairwise structural alignment with an SALIGN score of at least 30. The colored nodes represent proteins that are either a human solute carrier or similar in sequence to at least one human solute carrier. Nodes in white correspond to proteins that are not detectably similar in sequence to human solute carriers (i.e., an overlap of at least 150 residues of the target sequence to a sequence of a known structure at the sequence identity cutoff 20%, according to PSI-BLAST27).

Figure 2

Classification of solute carriers using similarity maps. The relationships between solute carrier sequences are visualized using the modified edge-weighted spring-embedded layout in cytoscape 2.6.1. Briefly, in the spring-embedded algorithm, the connected nodes are being attracted toward each other, while nodes that are unconnected are pushed apart. The nodes are connected by springs with resting lengths proportional to the shortest-path distance between them. The algorithm then iteratively adjusts the positions of each node to minimize the total “energy” of the system. The lengths of the springs are also determined by link weights, which are derived from the alignment scores better than a threshold. A: Each link represents a pairwise alignment with sequence identity of at least 25% and an E-value of less than 1. B: Each link represents a pairwise alignment with sequence identity of at least 10% and an E-value of less than 1.

Figure 3

Substrate type and transport mode mapped onto the solute carrier sequences. A: The colors represent the prototypical substrates of the transporters (see Materials and Methods section). In most cases, the sequence-based clustering also correlates with substrate type. For example, amino acid transporters, such as SLC6, SLC36, and SLC38, are clustered together (green). B: The colors represent the transport mode. The three major groups include cotransporters (yellow), exchangers (blue), and facilitators (red). “Orphan” nodes (cyan) represent transporters whose substrates are unknown, and “unknown” nodes (grey) represent transporters without a known transport mode. For some transporters, different modes of transport have been reported. For example, orange nodes mark transporters that are reported to be facilitators or cotransporters.

Figure 4

Conservation of solute carriers across eukaryotic organisms and tissues. A: The colors of the nodes indicate the oldest species in which the corresponding transporter is found, ranging from old (blue) to new (red). For instance, most members of the sugar transporter family SLC5 appeared for the first time in symmetrical organisms, whereas most SLC22 members appear only in higher mammals. B: The colors of the nodes indicate tissue specificity of the corresponding transporters, ranging from highly tissue-specific (red) to ubiquitous (blue). For example, SLC32 transporters, which are key proteins for synaptic release of inhibitory amino acids, are highly specific to the nervous system, whereas most members of the mitochondrial transporter family SLC25 are ubiquitously expressed.

Figure 5

Steps in identifying and classifying unknown solute carriers. (A) We extracted all human ORFs from Ensembl, (B) filtered out proteins with less than six predicted membrane α-helices, and (C) constructed multiple sequence profiles for the remaining sequences. (D) Simultaneously, a list of known solute carriers was extracted from public databases, and (E) again, for each protein sequence we created a multiple sequence profile. (F) We aligned a profile of each known solute carrier sequence with each of the human membrane protein profiles, resulting in a list of human membrane proteins that are similar to at least one known solute carrier. (G) Additional bioinformatics analysis, including construction of phylogenetic trees and detection of family-specific sequence motifs, allows us to identify high confidence predictions. (H) Finally, we verify our computational predictions experimentally by measuring the rate of substrate uptake into cells expressing tested transporters.

Figure 6

Predicted members of the SLC22 family. (A) Known SLC22 family members are represented by red nodes in the similarity network. An initial set of potential solute carriers is represented by pink nodes. Each link to these unannotated nodes indicates a pairwise alignment with sequence identity of at least 25% and an E-value of less than 1. The putative members of the SLC22 family were further evaluated using sequence analysis tools such as (B) phylogenetic trees and (C) analysis of family specific motifs. The SLC22 family is predicted to have two additional members [S22AX_HUMAN (ENS00000182157) and SVOPL_HUMAN] (black rectangles). A previous analysis of the SLC22 family also suggested that SVOPL might be an uncharacterized member.