We studied the phylogenetics and population genetics of CHC22 clathrin to understand the functional variation of this protein in relation to its evolutionary history. CHC22 clathrin is a key player in post-prandial blood glucose clearance in humans through its role in intracellular packaging of the GLUT4 glucose transporter in muscle and fat, the tissues in which CHC22 and GLUT4 are expressed (Vassilopoulos et al., 2009). The CHC22 pathway positions GLUT4 for cell surface release in response to insulin and consequent uptake of glucose into these tissues (Bryant et al., 2002). The CLTCL1 gene encoding CHC22 resulted from gene duplication that we have now dated to 494–451 MYA, early in vertebrate evolution when jawed vertebrates emerged. We had previously shown that CLTCL1 is a pseudogene in mice (Wakeham et al., 2005). Expanding analysis to 56 jawed vertebrate genomes (>5X coverage) we could not detect CLTCL1 in nine of them. Six of these absences can be ascribed to two independent gene loss events in branches of the Rodentia and the Cetartidactylae. The three others may represent additional gene losses or incomplete genome annotation. All vertebrate and non-vertebrate eukaryotes considered here have retained the parent CLTC gene encoding CHC17 clathrin, which mediates endocytosis and other housekeeping membrane traffic pathways. The analysis described here establishes that CLTC is under strong purifying selection. Notable is our evidence for purifying selection on CLTCL1 in the species in which it has been retained, supporting its functional importance in those species. Compared to CLTC, extensive allelic diversity was observed for CLTCL1 in all species for which populations were analyzed, including humans, chimpanzees and bears. Variant alleles were species-specific in most cases. In all human populations, two allelic variants of CLTCL1 are present in high frequency, differing only at one nucleotide, resulting in CHC22 protein with either methionine or valine at position 1316. The V1316 allotype appears specific to humans, but some non-human primates have a different variation at the position 1316. Analysis of ancient humans dated the appearance of the V1316 variant to 500–50 KYA and indicated that M1316, which is fixed in CHC17 clathrin, is the ancestral state. Analyses of human population genetic data provided support for the maintenance of high genetic diversity and two allotypes of CHC22. We hypothesize that selective pressure on CHC22 clathrin comes from its role in nutrient metabolism. Consistent with this hypothesis, we observed functional differences between the two CHC22 allotypes in their capacity to control GLUT4 membrane traffic, as predicted by structural modeling and differences in cellular dynamics of the two allotypes.

Retention of CLTC in all vertebrate species is consistent with the encoded CHC17 mediating cellular housekeeping clathrin functions shared by all eukaryotes. On the other hand, CHC22, encoded by the paralogous gene CLTCL1, operates in the specialized insulin-responsive GLUT4 pathway to make the pathway more efficient in those species that retained CLTCL1. Data presented here (Figure 8) and our recent mapping of a novel intracellular location for CHC22 function (Camus et al., 2018) indicate that, in human cells, CHC22 clathrin promotes transport from the secretory pathway to the insulin-responsive GSC. This CHC22 pathway complements the endocytic pathway for GLUT4 targeting to the GSC, so species without CHC22 can rely primarily on endocytosis for GLUT4 trafficking to the GSC, while species with CHC22 use both pathways. Thus, we hypothesize that species with functional CHC22 clathrin are more efficient at intracellular GLUT4 sequestration, resulting in lower surface GLUT4 in the absence of insulin, and tighter regulation of GLUT4 release in response to insulin. The trade-off is that these species have an inherent increased tendency to insulin resistance as their GLUT4 is sequestered more effectively. The two main vertebrate branches that have lost CHC22 comprise the Muridae (mice and rats) who are incessant herbivores and the Cetartiodactyla (sheep, cattle, porpoise and pigs) which include the ruminants (sheep and cattle) whose muscle uptake of glucose is critical for muscle function, but is not a main pathway for glucose clearance (Hocquette et al., 1995). These two groups of species require greater availability of GLUT4 on their cell surfaces, so that more efficient GLUT4 sequestration by CHC22 would not be favorable to their nutritional needs. The fact that CHC22 alters the balance of membrane traffic to the GSC means that species losing CLTCL1 could evolve compensatory pathways more compatible with their diets. Thus, transgenic mice expressing CHC22 over-sequester GLUT4 in their muscle and develop hyperglycemia with aging (Vassilopoulos et al., 2009). The cave fish, which appears to lack CLTCL1, has independently evolved mutations in the insulin receptor, creating natural insulin resistance, such that the presence of CHC22 on top of this mechanism might be detrimental (Riddle et al., 2018). The loss of CLTCL1 from cave fish is consistent with the insulin responsive GLUT4 pathway being a target for natural selection driven by diet, which might also explain CLTCL1 variation or loss for additional vertebrate species during vertebrate evolution.

The allelic variation reported here for CLTCL1 in human and bear populations further supports the hypothesis that CLTCL1 has undergone continued selection during vertebrate evolution in relation to diet. While purifying selection appears to be operating on CLTCL1 in those species that retain it, CLTCL1 is far more variable than CLTC in these species. In humans, we find two major and functionally distinct alleles at remarkably similar frequencies in all populations studied. Statistical analysis comparing early farmer and hunter-gatherer populations shows an apparent increase of the V1316 variant, suggesting a correlation with regular consumption of digestible carbohydrate. Notably, the SNP distinguishing these alleles is human-specific and likely arose 550–50 KYA (i.e. post-Neanderthal split, pre-Neolithic). Other dramatic increases in digestible carbohydrate utilization have been inferred for humans in this timeframe; in particular the advent of cooking (which gelatinizes crystalized starch, making it much easier to digest), salivary amylase gene copy number increase (allowing increased starch digestion capacity) and accelerated brain size increase (which would increase demands for blood glucose) (Hardy et al., 2015). While the co-evolution of these cultural and genetic traits was originally proposed to have occurred some 800 KYA, recent studies indicate a time frame of 450–300 KYA years for cooking (Shahack-Gross et al., 2014), increased oral amylase activity (Inchley et al., 2016) and accelerated brain size increase (Dunbar, 2019). The fact that the two major human CLTCL1 alleles are functionally distinct is consistent with diversifying selection operating on CLTCL1, with a balancing selection possibly caused by heterozygote advantage. While population genetic signatures for balancing or overdominant selection were not entirely robust, some summary statistics were suggestive of an increased diversity that was unlikely to have occurred under neutrality. Other statistics, such as the ones based on allele frequencies, would not be expected to gain significance within the timeframe of the human-specific diversifying selection we detect. The allelic diversity of CLTCL1 in other primate species could have the potential effect of diluting its function. Whilst chimpanzees are omnivores and gorillas herbivores, both rely for nutrition on extensive foraging for carbohydrate. Also notable is that polar bears, who have a very low carbohydrate diet compared to their brown bear relatives, have distinct CHC22 variants with unknown functionality, again consistent with CLTCL1 undergoing selection driven by nutritional ecology.

Clathrins are self-assembling proteins and function as a latticed network in the protein coat that they form on transport vesicles. Our structural modeling predicts that the single amino acid difference between the two main human CHC22 allotypes could influence the strength of molecular interactions in the CHC22 clathrin lattice, as position 1316 occurs at a lattice assembly interface (Figure 6). When expressed in cells, both CHC22 variants gave the same overall intracellular distribution, but CHC22-V1316 shows faster turnover from membranes than CHC22-M1316 (Figure 7) and is less effective at GLUT4 sequestration (Figure 8B). These properties are consistent with the methionine to valine change attenuating GLUT4 retention. This interpretation is further supported by a GLUT4 translocation assay, which indicates that the V1316 variant is less effective in forming the insulin-responsive GSC than the ancestral M1316 form of CHC22 (Figure 8A). Thus, mixed lattices occurring in heterozygous individuals, potentially reflect balancing selection and overdominance, might reduce GLUT4 sequestration compared to M1316 homozygotes. This would have the effect of improving glucose clearance. It can be argued that human consumption of digestible carbohydrate on a regular basis (Hardy et al., 2015), requiring increased glucose clearance, might be a selective force driving this genetic adaptation. This view is consistent with the increased frequency of the V1316 variant in early farmers. It is also possible that some forms of polar bear CHC22 are super-active at GLUT4 sequestration, providing a route to maintain high blood glucose, as occurs through other mutations in the cave fish (Riddle et al., 2018).

Regulators of fundamental membrane traffic pathways have diversified through gene duplication in many species over the timespan of eukaryotic evolution. Retention and loss can, in some cases, be correlated with special requirements resulting from species differentiation, such as the extensive elaboration of genes in the secretory pathway of Tetrahymena (Dacks and Robinson, 2017; Bright et al., 2010). The evolutionary history of CLTCL1, following vertebrate-specific gene duplication, suggests that differentiation of nutritional habits has shaped selection for the presence and absence of CLTCL1 in some vertebrate species, and its diversification in humans and potentially other species. Though its highest expression is in muscle and adipose tissue, transient expression of CHC22 during human brain development has also been documented (Nahorski et al., 2015). This was noted in a study of a very rare null mutant of CLTCL1 that caused loss of pain sensing in homozygotes and no symptoms for heterozygotes (Nahorski et al., 2015). Attenuated CHC22 function of the V1316 variant might lead to a spectrum of pain sensing in humans but this is unlikely to be a strong selective force affecting reproductive success, whereas glucose homeostasis, as suggested by our analysis, is more likely. By exerting efficient control of blood glucose levels, the presence of CHC22 clathrin was likely beneficial in providing the nutrition required to develop the large human brain, as well as affecting reproduction by influencing glucose availability during pregnancy (Hardy et al., 2015). However, over the last 12,500 years in association with farming, or perhaps over the last 450,000 years in association with cooking, salivary amylase activity and starch digestion (Hardy et al., 2015; Shahack-Gross et al., 2014; Inchley et al., 2016), readily available carbohydrate has increased our need to clear glucose from the blood, such that selection continues to act on CLTCL1 in humans. Our cell biology studies have also demonstrated that CHC22 increases GLUT4 retention. While we would not expect the major CLTCL1 polymorphism to directly influence the development of T2D, CHC22 accumulates on the expanded GSC that forms in cases of insulin-resistant T2D (Vassilopoulos et al., 2009), so its variation could potentially exacerbate insulin resistance to different degrees. The genetic diversity that we report here may reflect evolution towards reversing a human tendency to insulin resistance and have relevance to coping with increased carbohydrate in modern diets.