Evolution of the karyopherin-β family of nucleocytoplasmic transport factors; ancient origins and continued specialization

Abstract

Background: Macromolecular transport across the nuclear envelope (NE) is achieved through nuclear pore complexes (NPCs) and requires karyopherin-βs (KAP-βs), a family of soluble receptors, for recognition of embedded transport signals within cargo. We recently demonstrated, through proteomic analysis of trypanosomes, that NPC architecture is likely highly conserved across the Eukaryota, which in turn suggests conservation of the transport mechanisms. To determine if KAP-β diversity was similarly established early in eukaryotic evolution or if it was subsequently layered onto a conserved NPC, we chose to identify KAP-β sequences in a diverse range of eukaryotes and to investigate their evolutionary history.

Results: Thirty six predicted proteomes were scanned for candidate KAP-β family members. These resulting sequences were resolved into fifteen KAP-β subfamilies which, due to broad supergroup representation, were most likely represented in the last eukaryotic common ancestor (LECA). Candidate members of each KAP-β subfamily were found in all eukaryotic supergroups, except XPO6, which is absent from Archaeplastida. Phylogenetic reconstruction revealed the likely evolutionary relationships between these different subfamilies. Many species contain more than one representative of each KAP-β subfamily; many duplications are apparently taxon-specific but others result from duplications occurring earlier in eukaryotic history.

Conclusions: At least fifteen KAP-β subfamilies were established early in eukaryote evolution and likely before the LECA. In addition we identified expansions at multiple stages within eukaryote evolution, including a multicellular plant-specific KAP-β, together with frequent secondary losses. Taken with evidence for early establishment of NPC architecture, these data demonstrate that multiple pathways for nucleocytoplasmic transport were established prior to the radiation of modern eukaryotes but that selective pressure continues to sculpt the KAP-β family.

Figure 1. Schematic illustrating the basic functions of karyopherin-betas (KAP-βs) in context.

The nuclear envelope is punctuated by nuclear pores, within which sit the proteinaceous nuclear pore complexes. Transport is bidirectional via a central channel and is gated by an incompletely defined mechanism. KAP-βs participate in both import (blue panel) and export (pink panel), and are also known as importins and exportins respectively. However, many KAP-βs function in both modes and hence a clear designation between import and export is not apparent. Distinct cargo are imported and exported by formation of a complex in the origin compartment; this complex dissociates on reaching the destination compartment. The RanGTP/GDP gradient, which governs directionality of transport, is maintained by the localization of RanGEF to the nucleus and RanGAP to the cytosol. RanGDP is transported to the nucleus by its own import factor, Ntf2.

Figure 2. Neighbour-joining tree of KAP-β sequences across eukaryotes.

Six hundred and twenty two KAP-β candidate sequences, retrieved from 36 completed predicted proteomes, and representing five of six established eukaryotic supergroups, were clustered into a NJ tree with ClustalW. Taxa are coloured by species, listed on right, and by eukaryotic supergroup. All sequences highlighted by a black arc at the rim of the tree exhibit evidence for specific KAP-β subfamily membership and are located on a branch immediately adjacent to at least one other similar taxon on the tree. Unhighlighted sequences either have some evidence for sub-family membership but are not clustered, or are orphans. The subfamily name of each cluster is followed by additional names, based upon S. cerevisiae and H. sapiens gene names . Tree drawn using PhyloWidget .

Figure 3. Phylogeny of selected representatives of the fifteen KAP-β subfamilies.

Numbers on internodes refer to PhyML bootstrap support/MrBayes posterior probability values and the MrBayes topology is shown. Dots indicate values better than 75% bootstrap support and 0.95 posterior probability, while full values are given for important internodes supporting KAP subfamilies. The colour scheme is as in figure 2 and species included are as follows: Homo sapiens (HUMAN), Nematostella vectensis (Nemve), Phytophthora ramorum (Phyra), Phytophthora sojae (Physo), Arabidopsis thaliana (ARATH) and Physcomitrella patens (Phypa). Subfamilies are indicated by vertical bars and inidvidual sequences are represented by gene IDs.

Figure 4. Unrooted karyopherin-β subfamily phylogeny.

Schematic illustrating inferred ancestral relationships between the KAP-β subfamilies, percent identity (%id) values, known roles as import or export factor (I/E) within each subfamily and description of cargo types. This unrooted topology was inferred from a series of phylogenetic reconstructions available in Figure S1. Colored panels highlight three clades of related subfamilies whose phylogenies were initially determined; a subfamily representative of each of these clades, and of XPO6, were then used to infer a family-wide phylogeny.

Figure 5. Subfamily distribution of karyopherin-βs across the Eukaryota.

Black circles indicate presence of a phylogenetically supported (see methods) KAP-β subfamily member. Grey circles indicate candidate subfamily members that could not be verified phylogenetically. Empty circles indicate no candidate found. Numbered circles indicate cases where more than one candidate is found. A small circle indicates candidate(s) in addition to phylogenetically supported candidate(s) indicated by big circles. The left panel illustrates the phylogenetic relationships between subfamilies. See Table S1 for additional information including protein identifiers.

Figure 6. Schematic illustrating lineage-specific events in KAP-β family evolution.

Proposed positions of origin and secondary loss are shown on a schematic eukaryotic phylogeny, representing five major supergroups. Dots indicate expansions and losses; note the position in an internode is arbitrary, and only events that are shared by more than one taxon are shown. TNPO3 is proposed to have undergone an ancestral duplication (A and B) followed by loss in the lineage leading to the modern Excavata. Note that the more recently accepted SAR supergroup, encompasing stramenopiles, alveolates and Rhizaria is used here. Figure adapted from .