Widely Distributed Proteins Homologous to Vertebrate Metaxins Implicated in Protein Import Into Mitochondria

bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.895979; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Invertebrate Metaxins 1 and 2: Widely Distributed Proteins Homologous to Vertebrate Metaxins Implicated in Protein Import into Mitochondria

Kenneth W. Adolph

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455

Email Address: [email protected]

ABSTRACT

Metaxin 1 and 2 genes, previously investigated in vertebrates, are shown to be widely

distributed among invertebrates. But metaxin 3 is absent. The predicted proteins of the

invertebrate metaxins were initially identified by homology with human metaxin 1 and 2 proteins,

and by the presence of characteristic GST_Metaxin protein domains. Invertebrate metaxins were

revealed for a variety of phyla, including Echinodermata, Cnidaria, Porifera, Chordata,

Arthropoda, Mollusca, Brachiopoda, Placozoa, and Nematoda. Metaxins were also found in

insects (Arthropoda) of different taxonomic orders: Diptera, Coleoptera, Lepidoptera,

Hymenoptera, and Blattodea. Invertebrate and human metaxin 1 proteins have about 41% identical

amino acids, while metaxin 2 proteins have about 49% identities. Invertebrate and vertebrate

metaxins share the same characteristic protein domains, further strengthening the identification of

the invertebrate proteins as metaxins. The domains are, for metaxin 1, GST_N_Metaxin1_like,

GST_C_Metaxin1_3, and Tom37. For metaxin 2, they are GST_N_Metaxin2, GST_C_Metaxin2,

and Tom37. Phylogenetic trees show that invertebrate metaxin 1 and metaxin 2 proteins are

related, but form separate groups. The invertebrate proteins are also closely related to vertebrate

metaxins, though forming separate clusters. These phylogenetic results indicate that all metaxins

likely arose from a common ancestral sequence. The neighboring genes of the invertebrate

metaxin 1 and 2 genes are largely different for different invertebrate species. This is unlike the

situation with vertebrate metaxin genes, where, for example, the metaxin 1 gene is adjacent to the

thrombospondin 3 gene. The dominant secondary structures predicted for the invertebrate

metaxins are alpha-helical segments, with little beta-strand. The conserved pattern of helical

segments is the same as that found for vertebrate metaxins 1, 2, and 3.

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1. INTRODUCTION

The metaxins have previously been identified as vertebrate proteins involved in the import

of newly synthesized proteins into mitochondria. Three vertebrate metaxins have been

characterized: metaxin 1 (Bornstein et al., 1995), metaxin 2 (Armstrong et al., 1999), and metaxin

3 (Adolph, 2004, 2005). The vertebrate metaxins are thought to be components of protein

complexes functionally equivalent to the TOM or SAM complexes of the outer mitochondrial

membrane of yeast (Saccharomyces cerevisiae). The role of the TOM and SAM complexes in

protein import into yeast mitochondria is much better understood than protein import in human

cells and other vertebrate cells. This investigation concerns whether invertebrates, like vertebrates,

have metaxin genes. The aims have been to understand which, if any, of the metaxins are found in

invertebrates, how widely distributed they might be among invertebrate phyla, and, most

importantly, fundamental properties of the metaxins encoded by invertebrate genomes. The results

show that metaxin 1 and 2 genes are widely distributed among invertebrates, just as in vertebrates.

But although metaxin 3 is highly conserved among vertebrates (Adolph, 2019), metaxin 3 was not

found for the invertebrates studied.

The invertebrates in this investigation represent many of the invertebrate phyla. An

emphasis was on invertebrates that are widely used as model organisms in biological and

biomedical research. These include the classic model organisms C. elegans (Brenner, 1974; White

et al., 1986) and Drosophila melanogaster (Wangler et al., 2017). Both had their genomes

sequenced early in the history of whole genome sequencing (C. elegans sequencing consortium,

1998; Adams et al., 2000). Other organisms in this study that are important model systems and

have had their genomes sequenced include Ciona intestinalis (Satoh, 2003; Dehal et al., 2002),

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Nematostella vectensis (Genikhovich and Technau, 2009; Putnam et al., 2007), and Trichoplax

adhaerens (Schierwater and DeSalle, 2018; Srivastava et al., 2008).

The first metaxin gene to be identified was the mouse metaxin 1 gene (Mtx1) (Bornstein

et al., 1995). The metaxin 1 protein was revealed to be located in the outer mitochondrial

membrane, and experimental evidence demonstrated a role in protein import into mitochondria

(Armstrong et al., 1997). The mouse metaxin 1 gene is in close proximity to the thrombospondin 3

gene (Thbs3) and the glucocerebrosidase gene (Gba). The thrombospondins are extracellular

matrix proteins, and are important mediators of cell-matrix and cell-cell interactions (Adams and

Lawler, 2011; Mosher and Adams, 2012).

A gene homologous to the mouse metaxin 1 gene was found to be present in humans at

cytogenetic location 1q21. In human cells, the metaxin 1 gene (MTX1) is between a

glucocerebrosidase pseudogene (psGBA1) and the gene for thrombospondin 3 (THBS3) (Long et

al., 1996; Adolph et al., 1995), with a metaxin 1 pseudogene (psMTX1) also at this locus. The gene

order is GBA1—psMTX1—psGBA1—MTX1—THBS3. In humans, a deficiency of the

glucocerebrosidase enzyme as a result of mutations in GBA1 can cause Gaucher disease (Mistry et

al., 2017; Tayebi et al., 2003; LaMarca et al., 2004). An increased genetic risk for the development

of Parkinson’s disease is also associated with GBA1 mutations (Do et al., 2019).

A second metaxin protein was identified in the mouse through its interaction with metaxin

1 protein (Armstrong et al., 1999). The metaxin 2 protein shows 29% amino acid identities with

mouse metaxin 1. The gene (Mtx2) is adjacent to a group of homeobox genes coding for

transcription factors. Because of their interaction, metaxin 1 and metaxin 2 proteins might form a

complex in the outer mitochondrial membrane involved in the import of proteins into

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mitochondria. A human gene (MTX2) homologous to Mtx2 is at 2q31.2. Like the mouse gene, it is

adjacent to a cluster of homeobox genes.

Zebrafish metaxin 3 was the first metaxin 3 to be identified (Adolph, 2004). The zebrafish

metaxin 3 protein sequence, deduced from its cDNA sequence, was 40% homologous to zebrafish

metaxin 1, and 26% homologous to metaxin 2. Xenopus laevis metaxin 3 was also identified

through sequencing of cDNAs (Adolph, 2005). Zebrafish and Xenopus metaxin 3 proteins have

55% amino acid identities, indicating that the proteins are conserved to a great degree. The

recognition that metaxin 3 is a metaxin but distinct from metaxins 1 and 2 was based on factors

including amino acid sequence homology, presence of GST_Metaxin domains, phylogenetic

analysis, and conserved patterns of protein secondary structure.

The import of proteins into mitochondria has been investigated in great detail in yeast

(Saccharomyces cerevisiae) (Pfanner et al., 2019; Wiedemann and Pfanner, 2017; Neupert, 2015).

In particular, the TOM complex (translocase of the outer mitochondrial membrane complex) and

SAM complex (sorting and assembly machinery complex) bring about the import of beta-barrel

proteins into yeast mitochondria and their insertion into the outer membrane. The Tom37 domain,

a conserved protein domain of the SAM complex, is also present in the metaxin proteins, both

vertebrate and invertebrate (this study).

Although the invertebrates include important research organisms used in many labs,

nothing has been published about the metaxins of invertebrates. Metaxin proteins representing

diverse invertebrate phyla have therefore been studied. The proteins were primarily those

predicted by genome sequencing. Metaxin 1 and metaxin 2 were found to be highly conserved

among invertebrates, and fundamental properties of the proteins were determined.

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2. MATERIALS AND METHODS

The amino acid sequence alignments in Figure 1 were produced using the Global Align

program of the NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi; Needleman and Wunsch, 1970;

Altschul et al., 1997). This program allowed the identities and similarities of two metaxin amino

acid sequences to be determined along their entire lengths. The EMBOSS Needle program

(https://www.ebi.ac.uk/Tools/psa/emboss_needle/) was also used to generate pairwise sequence

alignments. The CD_Search tool (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi; Marchler-

Bauer et al., 2017) was used to identify the protein domains included in Figure 2 through

searching the conserved domain database of the NCBI. Evolutionary relationships of invertebrate

metaxins (Figure 3) were examined with the COBALT multiple sequence alignment tool and the

phylogenetic trees produced from the multiple sequence alignments

(www.ncbi.nlm.nih.gov/tools/cobalt; Papadopoulos and Agarwala, 2007). In addition, the Clustal

Omega program (https://www.ebi.ac.uk/Tools/msa/clustalo/) was used to generate multiple

sequence alignments and phylogenetic trees. The genes adjacent to invertebrate metaxin genes

were identified from the related information on “Gene neighbors” that resulted from BLAST

searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Adjacent genes were also identified using the

Genome Data Viewer and, earlier, the Map Viewer genome browsers

(www.ncbi.nlm.nih.gov/genome/gdv). The PSIPRED secondary structure prediction server

(bioinf.cs.ucl.ac.uk/psipred/; Jones, 1999) was used to predict the alpha-helical structures of

invertebrate metaxin proteins in Figure 4. The presence of transmembrane helices was investigated

with the PHOBIUS program (www.ebi.ac.uk/Tools/pfa/phobius/; Madeira et al., 2019) and also

the TMHMM prediction server (www.cbs.dtu.dk/services/TMHMM/; Krogh et al., 2001).

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3. RESULTS AND DISCUSSION

3.1. Conservation of Metaxin Proteins Among Invertebrates

In this investigation, invertebrates were found to have distinct metaxin 1 and metaxin 2

genes like humans and other vertebrates. However, invertebrates were not observed to have

metaxin 3 genes. Plants and bacteria are different and have a single metaxin-like gene (K.W.

Adolph, unpublished). For invertebrates other than insects, metaxin 1 and 2 genes were found for

representative invertebrates in a variety of phyla. These phyla include Echinodermata (example:

sea urchin), Cnidaria (coral), Porifera (sponge), Chordata (amphioxus), Arthropoda (spider),

Mollusca (octopus), Brachiopoda (brachiopod), Placozoa (Tricoplax), and Nematoda (C. elegans).

Insects were also found to have metaxin 1 and metaxin 2 genes, while metaxin 3 was not

observed. This was true for representative insects (Phylum: Arthropoda; Class: Insecta) of various

taxonomic orders: Diptera (example: fruit fly), Coleoptera (beetle), Lepidoptera (moth),

Hymenoptera (honey bee), Hemiptera (bed bug), and Blattodea (termite).

For the fruit fly Drosophila, a second metaxin 2 gene was present in 13 of 17 Drosophila

species studied. The two genes are designated metaxins 2a and 2b. The predicted 2a and 2b

proteins have about 48% sequence identities. However, other insects have a single metaxin 2 gene,

including the Mediterranean fruit fly, oriental fruit fly, and olive fruit fly. Different species of

Drosophila have very similar metaxin proteins. For example, metaxin 1 proteins of different

species have about 85% sequence identities, and metaxin 2a proteins 91%.

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3.2. Amino Acid Sequence Homology of Invertebrate Metaxins

Invertebrate metaxin genes and proteins were first identified by homology with the amino

acid sequences of human metaxin 1 and human metaxin 2 proteins. The invertebrate protein

sequences are, in most cases, the sequences predicted from genomic DNA sequences. The

identification of the invertebrate proteins as metaxins was strengthened by the presence of

conserved, characteristic protein domains, the GST_N_Metaxin, GST_C_Metaxin, and Tom37

domains, but no additional major domains. Also, the lengths of the invertebrate metaxin proteins

had to be comparable to human metaxin 1 (317 amino acids) or human metaxin 2 (263 amino

acids).

The invertebrate metaxin 1 proteins share about 41% amino acid sequence identities with

human metaxin 1. The metaxin 2 proteins share about 49% identities with human metaxin 2. As an

example, the metaxin 1 protein sequence of the purple sea urchin Strongylocentrotus purpuratus

(Echinodermata) has 46% identical amino acids when aligned with human metaxin 1. As

additional examples, the sponge Amphimedon queenslandica (Porifera) has 28% identities, and the

Pacific oyster Crassostrea gigas (Mollusca) has 39% identities. When aligned with the human

metaxin 2 protein sequence, the sea urchin metaxin 2 sequence shows 50% identities, the sponge

metaxin 2 sequence 35%, and the oyster sequence 47%.

Figure 1(A) includes the sequence of human metaxin 1 aligned with that of the roundworm

C. elegans (Caenorhabditis elegans; Nematoda). There are 31% amino acid identities. In Figure

1(B), 36% identities are found in aligning human metaxin 2 with C. elegans metaxin 2. C.elegans

is extensively used as a model organism for investigating the genetic control of development and

physiology (Brenner, 1974; White et al., 1986), and was the first multicellular organism to have its

genome completely sequenced (C. elegans sequencing consortium, 1998).

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Figure 1(C) shows the alignment of human metaxin 1 with Drosophila melanogaster

metaxin 1. The identities are 27%. For human and Drosophila metaxin 2a, 41% identities are

observed (Figure 1(D)). The Drosophila metaxins are included because Drosophila melanogaster

is widely used as a model organism for studies of molecular genetics, population genetics, and

developmental biology (Adams et al., 2000; Wangler et al., 2017).

3.3. Protein Domains of Invertebrate Metaxins

A defining characteristic of metaxin proteins is the presence of GST_N_Metaxin and

GST_C_Metaxin domains, and, in addition, Tom37 domains. Figure 2(A) shows the domain

structure of representative invertebrate metaxin 1 and metaxin 2 proteins, while insects are in

Figure 2(B). The invertebrates in 2(A) include the sea squirt Ciona intestinalis, which has the

smallest genome of any chordate that can be utilized experimentally. As shown in the figure,

Ciona metaxin 1 has GST_N_Metaxin1_like, GST_C_Metaxin1_3, and Tom37 domains. Ciona

metaxin 2 has GST_N_Metaxin2, GST_C_Metaxin2, and Tom37 domains. The metaxin domains

of the starlet sea anemone (Nematostella vectensis) and Trichoplax adhaerens are also included in

Figure 2(A). The domains are identical to those of Ciona. The starlet sea anemone is a Cnidarian,

the simplest animals with cells organized into tissues. Trichoplax adhaerens is the simplest known

animal and has the smallest known genome of any animal. (For references, see Introduction.) The

metaxin 1 and metaxin 2 domain structures of the three organisms are seen to be highly conserved.

The metaxin proteins of vertebrates, in particular humans, have similar domain structures

to those seen in Figure 2. Human metaxin 1 and metaxin 3 have GST_N_Metaxin1_like,

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GST_C_Metaxin1_3, and Tom37 domains. Human metaxin 2 has GST_N_Metaxin2,

GST_C_Metaxin2, and Tom37 domains.

The metaxin domains of representative insects are shown in Figure 2(B). The metaxin 1

and metaxin 2 domains are the same as those in 2(A) for invertebrates that don’t include insects.

The examples in Figure 2(B) are the yellow fever mosquito (Aedes aegypti), a dampwood termite

(Zootermopsis nevadensis), and the common bed bug (Cimex lectularius).

The Tom37 domain, as identified by the NCBI conserved domain database, is present in

yeast (Saccharomyces cerevisiae) protein Tom37, now generally known as Sam37. Sam37 is a

component of the SAM complex, the sorting and assembly machinery complex. The SAM

complex, consisting of Sam50, Sam37, Sam35, and Mdm10, is located in the outer mitochondrial

membrane, and is involved in the insertion of beta-barrel proteins into the outer membrane. The

involvement of Sam37 (Tom37) in protein import into mitochondria of yeast is suggestive of a

similar role for the metaxins in vertebrates and invertebrates, and such a role in human and mouse

mitochondria has been experimentally demonstrated.

3.4. Evolutionary Relationships of Invertebrate Metaxin Proteins

Phylogenetic analysis demonstrates that the invertebrate metaxin 1 and metaxin 2

homologs are grouped separately from vertebrate metaxins, including human metaxins. Figure

3(A) shows the phylogenetic relationships of selected invertebrate and vertebrate metaxins. The

analysis was based on multiple sequence alignments of the protein sequences. The horizontal

lengths of the branches are proportional to the extent of evolutionary change. As Figure 3(A)

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demonstrates, the metaxin 1 proteins form a distinct group relative to the metaxin 2 proteins. And

within each group, the invertebrate and vertebrate proteins form separate clusters.

Also within each group, invertebrate metaxins of the same phylum are seen to be the most

closely related. This includes the starfish and sea urchin metaxin 1 and metaxin 2 proteins

(Echinodermata), the oyster and snail metaxins (Mollusca), and the horseshoe crab and scorpion

metaxins (Arthropoda). Similarly, for vertebrate metaxin 1 and metaxin 2 proteins, the human and

mouse proteins are more closely related compared to the Xenopus and zebrafish metaxins.

The observed grouping of the invertebrate and vertebrate metaxin 1 proteins indicates that

the metaxin 1 proteins arose from a common ancestor. The separate grouping of the metaxin 2

proteins likewise indicates that the metaxin 2 proteins originated from a common ancestor. In

addition, the results strengthen the likelihood of a common ancestral gene sequence for both of the

metaxins. This is true even though alignments of the metaxin 1 and 2 protein sequences show

relatively low percentages of amino acid identities (22% for human metaxins 1 and 2).

Insect metaxins are included in Figure 3(B). As with the other invertebrate metaxins in

Figure 3(A), the insect metaxin 1 and metaxin 2 proteins form separate groups. The metaxins of

insects of the same taxonomic order are seen to be more closely related than those in different

orders. The examples shown are moth and silkworm metaxins (Order: Lepidoptera), honey bee

and bumblebee metaxins (Hymenoptera), and Drosophila metaxins of different species (Diptera).

Although two distinct groupings are found for the metaxin 1 and metaxin 2 proteins, the insect

metaxins, like other invertebrate metaxins, most likely originated from an ancestral metaxin, even

though the amino acid sequences of metaxins 1 and 2 have diverged significantly.

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The metaxins of the three species of Drosophila in Figure 3(B) are very closely related

phylogenetically. For metaxin 2, the two Drosophila proteins (metaxins 2a and 2b) found for a

number of Drosophila species are divided into a metaxin 2a cluster and a metaxin 2b cluster. The

results indicate that the 2a proteins are related through a common ancestor, and similarly for the

2b cluster. And both the 2a and 2b proteins are likely to have originated from a common metaxin 2

ancestral sequence.

3.5. Neighboring Genes of Invertebrate Metaxins

The genes adjacent to invertebrate metaxin 1 and metaxin 2 genes are largely different

comparing one invertebrate with another. Also, no similarities are seen with the arrangement of

genes in humans and other vertebrates, where the metaxin 1 gene (MTX1) is next to the

thrombospondin 3 gene (THBS3), the metaxin 2 gene (MTX2) is adjacent to a cluster of homeobox

genes, and the metaxin 3 gene (MTX3) is next to the thrombospondin 4 gene (THBS4). In contrast,

invertebrate metaxin genes are not adjacent to thrombospondin or homeobox genes. And the genes

in the chromosomal regions up to 10 to 20 or more genes from the metaxin genes are different

than the genes of humans and other vertebrates, and generally differ from one invertebrate to

another. For example, the roundworm C. elegans, which has a well-annotated genome, has a

metaxin 1 gene (mtx-1) between the humpback gene and a heat-shock protein gene. And a metaxin

2 gene (mtx-2) is next to the prolyl carboxy peptidase gene. Unique arrangements of genes

adjacent to the metaxin genes were also found for Ciona intestinalis, a sea squirt, with the metaxin

1 gene (mtx-1) between genes for a regulatory protein and a transforming, acidic coiled-coil

protein. There are exceptions, however, to having different neighboring genes. A few genes were

found to be close to the metaxin genes of some, but not all, invertebrates. As an example, an ADP-

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dependent glucokinase gene is close to 3 of 10 invertebrate metaxin 1 genes. But the metaxin 2

genes of the 10 invertebrates showed no similarities in adjacent genes.

More specifically for insects, the genes near metaxin 1 genes are different for Drosophila

melanogaster compared to the Mediterranean fruit fly, honey bee, etc. Also, for Drosophila

melanogaster, the genes close to the two metaxin 2 genes (2a and 2b) are different. However, the

genes adjacent to the single metaxin 1 homolog are conserved in ten of twelve Drosophila species.

Other exceptions to having different genes are closely related species such as the oriental fruit fly

and olive fruit fly, which have the same 5 adjacent genes.

3.6. Secondary Structures of Invertebrate Metaxin Proteins

Alpha-helical segments are the dominant secondary structures of invertebrate metaxins.

There is relatively little beta-strand. Figure 4 shows that representative invertebrate metaxin 1 and

2 proteins each have eight predicted helical segments (H2 through H9) with the same pattern of

spacing between the segments. Metaxin 1 proteins have an additional 9th helical segment (H10) in

their extended C-terminal regions, while metaxin 2 proteins have a 9th helix (H1) in their N-

terminal regions. This pattern of alpha-helices is the same as the pattern for metaxins 1, 2, and 3 of

humans and other vertebrates. As shown in Figure 4(A), the conserved pattern of helices was

found for invertebrate phyla including Cnidaria (example: coral), Porifera (sponge), Arthropoda

(amphipod), Mollusca (octopus), Chordata (amphioxus), and Brachiopoda (brachiopod). Figure

4(B) includes multiple sequence alignments showing the pattern of conserved helices for a

selection of insects (Phylum: Arthropoda; Class: Insecta). The insect taxonomic orders represented

in Figure 4(B) are Hymenoptera (ant, bumblebee), Coleoptera (beetle), Diptera (fruit fly),

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Blattodea (termite), and Hemiptera (whitefly). The pattern of predicted alpha-helical segments is

very similar to the pattern in Figure 4(A) for representative invertebrates, not including insects.

Eight helices (H2 to H9) are found for both metaxin 1 and metaxin 2 of insects. H1, an extra N-

terminal helix, is characteristic of metaxin 2 proteins. H10, an additional C-terminal helix,

characterizes metaxin 1 proteins. These highly conserved patterns are found for other insects and

non-insect invertebrates not included in Figure 4.

Metaxin 1 proteins of vertebrates have a predicted transmembrane helix near the C-

terminus. However, vertebrate metaxin 2 and metaxin 3 proteins don’t have a C-terminal

transmembrane helix. The metaxin 1 transmembrane helix is identified as helix H10 of

vertebrates, since it largely coincides with H10. The transmembrane helix could serve to anchor

metaxin 1 proteins to the outer mitochondrial membrane in connection with their role in protein

import into mitochondria. The presence of a C-terminal transmembrane helix is also predicted for

many invertebrate metaxin 1 proteins. For example, the sea urchin Strongylocentrotus purpuratus

has a transmembrane helix between residues 272 and 294 of the 319 amino acid protein. Belcher’s

lancelet or amphioxus metaxin 1 has the helix between residues 269 and 291 of the 321 amino

acid protein. The transmembrane helix mostly coincides with C-terminal helix H10. But neither of

the metaxin 2 proteins of these invertebrates has a transmembrane helix. Considering insects, the

Drosophila melanogaster metaxin 1 homolog (CG9393) has a transmembrane helix between

residues 281 and 301 of the 316 amino acid protein. But neither of the two D. melanogaster

metaxin 2 homologs (CG5662 and CG8004) has a transmembrane helix.

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15 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.895979; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Bornstein, P., McKinney, C.E., LaMarca, M.E., Winfield, S., Shingu, T., Devarayalu, S., Vos, H.L. and Ginns, E.I., Metaxin, a gene contiguous to both thrombospondin 3 and glucocerebrosidase, is required for embryonic development in the mouse: implications for Gaucher disease. Proc. Natl. Acad. Sci. USA 92, 4547-4551 (1995). Brenner, S., The genetics of Caenorhabditis elegans. Genetics 77, 71-94 (1974). C. elegans sequencing consortium, Genome sequence of the nematode C. elegans: a platform for investigating biology, Science 282, 2012-2018 (1998). Dehal, P., Satou, Y., Campbell, R.K. et al., The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science 298, 2157-2167 (2002). Do, J., McKinney, C., Sharma, P. and Sidransky, E., Glucocerebrosidase and its relevance to Parkinson disease. Mol. Neurodegener. 14, 36 (2019) doi:10.1186/s13024-019-0336-2. Genikhovich, G. and Technau, U., The starlet sea anemone Nematostella vectensis: an anthozoan model organism for studies in comparative genomics and functional evolutionary developmental biology. Cold Spring Harb. Protoc. (2009) pdb.emo129. doi: 10.1101/pdb.emo129 Jones, D.T., Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292, 195-202 (1999). Krogh, A., Larsson, B., von Heijne, G. and Sonnhammer, E.L.L., Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567-580 (2001). LaMarca, M.E., Goldstein, M., Tayebi, N., Arcos-Burgos, M., Martin, B.M. and Sidransky, E., A novel alteration in metaxin 1, F202L, is associated with N370S in Gaucher disease. J. Hum. Genet. 49, 220-222 (2004). Long, G.L., Winfield, S., Adolph, K.W., Ginns, E.I. and Bornstein, P., Structure and organization of the human metaxin gene (MTX) and pseudogene. Genomics 33, 177-184 (1996).

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Madeira, F., Park, Y.M., Lee, J., Buso, N., Gur, T., Madhusoodanan, N., Basutkar, P., Tivey, A.R.N., Potter, S.C., Finn, R.D. and Lopez, R., The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 47, W636-W641 (2019). Marchler-Bauer, A., Bo, Y., Han, L., He, J., Lanczcki, C.J., Lu, S., Chitsaz, F., Derbyshire, M.K., Geer, R.C., Gonzales, N.R., Gwadz, M., Hurwitz, D.I., Lu, F., Marchler, G.H., Song, J.S., Thanki, N., Wang, Z., Yamashita, R.A., Zhang, D., Zheng, C., Geer, L.Y. and Bryant, S.H., CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 45, D200-D203 (2017). Mistry, P.K., Lopez, G., Schiffmann, R., Barton, N.W., Weinreb, N.J. and Sidransky, E., Gaucher disease: progress and ongoing challenges. Mol. Genet. Metab. 120, 8-21 (2017). Mosher, D.F. and Adams, J.C., Adhesion-modulating/matricellular ECM protein families: a structural, functional and evolutionary appraisal. Matrix Biol. 31, 155-161 (2012). Needleman, S.B. and Wunsch, C.D., A general method applicable to the search for similarities in the amino acid sequences of two proteins. J. Mol. Biol. 48, 443-453 (1970). Neupert, W., A perspective on transport of proteins into mitochondria: a myriad of open questions. J. Mol. Biol. 427, 1135-1158 (2015). Papadopoulos, J.S. and Agarwala, R., COBALT: constraint-based alignment tool for multiple protein sequences. Bioinformatics 23, 1073-1079 (2007). Pfanner, N., Warscheid, B. and Wiedemann, N., Mitochondrial proteins: from biogenesis to functional networks. Nat. Rev. Mol. Cell Biol. 20, 267-284 (2019). Putnam, N.H., Srivastava, M., Hellsten, U. et al., Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317, 86-94 (2007). Satoh, N., The ascidian tadpole larva: comparative molecular development and genomics. Nat. Rev. Genet. 4, 285-295 (2003). Schierwater, B. and DeSalle, R., Placozoa. Curr. Biol. 28, R97-R98 (2018). Srivastava, M., Begovic, E., Chapman, J. et al., The Trichoplax genome and the nature of placozoans. Nature 454, 955-960 (2008).

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Tayebi, N., Stubblefield, B.K., Park, J.K., Orvisky, E., Walker, J.M., LaMarca, M.E. and Sidransky, E., Reciprocal and nonreciprocal recombination at the glucocerebrosidase gene region: implications for complexity in Gaucher disease. Am J. Hum. Genet. 72, 519-534 (2003). Wangler, M.F., Yamamoto, S., Chao, H.T., Posey, J.E., Westerfield, M., Postlethwait, J., Hieter, P., Boycott, K.M., Campeau, P.M. and Bellen, H.J., Model organisms facilitate rare disease diagnosis and therapeutic research. Genetics 207, 9-27 (2017). White, J.G., Southgate, E., Thomson, J.N. and Brenner, S., The structure of the nervous system of the nematode Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 314, 1-340 (1986). Wiedemann, N. and Pfanner, N., Mitochondrial machineries for protein import and assembly. Annu. Rev. Biochem. 86, 685-714 (2017).

18 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.895979; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

FIGURE LEGENDS

Figure 1. Homology of invertebrate metaxins: amino acid sequence alignments. (A) The

amino acid sequence of human metaxin 1 (317 amino acids) is aligned with the sequence of a

representative invertebrate C. elegans (312 aa), an important model system for studies of

development. The identities and similarities are shown in red between the sequences. Identities are

31% and similarities are 46% for this example. Invertebrates, generally, share about 41% amino

acid sequence identities with human metaxin 1. (B) The alignment of human metaxin 2 (263 aa)

with C. elegans metaxin 2 (244 aa) shows 36% identical amino acids, with 57% similarities.

Invertebrate metaxin 2 sequences more typically share about 49% identities relative to human

metaxin 2. Comparing the sequences of various invertebrate metaxin 1 proteins with invertebrate

metaxin 2 proteins shows about 27% identities. (C) Alignment of human metaxin 1 and an insect

metaxin 1. In this alignment with a Drosophila melanogaster metaxin 1 homolog (315 aa), 27%

identical amino acids and 45% similarities are found. D. melanogaster was included because it is a

classic model system of genetics and developmental biology. Comparing the metaxin 1 sequences

of insects of different orders with each other shows about 38% identities. (D) For human metaxin

2 and D. melanogaster metaxin 2a (269 aa), there are 41% identities and 61% similarities. D.

melanogaster is one of the Drosophila species with two metaxin 2 proteins, 2a and 2b. The two

proteins have 48% sequence identities. Comparing the metaxin 2 sequences of a variety of insects

of different orders shows about 51% homology.

Figure 2. Domain structure of invertebrate metaxin proteins. (A) The metaxin 1 and metaxin 2

protein domains of representative invertebrates, not including insects, are shown. These are the

characteristic GST_N_Metaxin, GST_C_Metaxin, and Tom37 domains. The invertebrate

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examples include a sea squirt (Ciona intestinalis; Phylum: Chordata), a sea anemone

(Nematostella vectensis; Phylum: Cnidaria), and Tricoplax adhaerens (Phylum: Placozoa). The

domain structures are highly conserved in these examples, and in the metaxins of other

invertebrates. (B) The metaxin protein domains are shown for three representative insects: the

yellow fever mosquito (Aedes aegypti; Order: Diptera), a termite (Zootermopsis nevadensis;

Order: Blattodea), and the common bed bug (Cimex lectularius; Order: Hemiptera). Other insects

have the same domains, and the insect domains are the same as for the invertebrates in (A). The

presence of a Tom37 domain in both metaxin 1 and 2 proteins is in keeping with a possible role

for the invertebrate metaxins in the import of proteins into mitochondria. Tom37 is a yeast protein

with a well-characterized role in protein import.

Figure 3. Evolutionary relationships of metaxin 1 and 2 proteins of invertebrates.

(A) Phylogenetic tree of selected invertebrate and vertebrate metaxins. The invertebrate species

shown are the crown-of-thorns starfish (Acanthaster planci), purple sea urchin (Strongylocentrotus

purpuratus), Pacific oyster (Crassostrea gigas), snail (Biomphalaria glabrata), Atlantic horseshoe

crab (Limulus polyphemus), and bark scorpion (Centruroides sculpturatus). The starfish and sea

urchin are examples of the phylum Echinodermata, the oyster and snail are Mollusca, and the

horseshoe crab and scorpion are Arthropoda. (B) The insect metaxins are those of a moth

(Amyelois transitella), the domestic silkworm (Bombyx mori), the Western honey bee (Apis

mellifera), and the common eastern bumblebee (Bombus impatiens). Three Drosophila species are

also included (Drosophila melanogaster, Drosophila elegans, and Drosophila eugracilis). The

moth and silkworm are examples of the taxonomic order Lepidoptera, the honey bee and

bumblebee are Hymenoptera, and the Drosophila species are Diptera.

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Figure 4. Secondary structures of invertebrate metaxin proteins: multiple sequence

alignments. (A) Representative invertebrate metaxin 1 and metaxin 2 amino acid sequences are

aligned, and the predicted alpha-helical segments are underlined and shown in red. The helices are

labeled H1 through H10. The invertebrate species include a stony coral (Acropora digitifera;

Phylum: Cnidaria), a sponge (Amphimedon queenslandica; Porifera), an amphipod crustacean

(Hyalella azteca; Arthropoda), the California two-spot octopus (Octopus bimaculoides; Mollusca),

the Florida lancelet or amphioxus (Branchiostoma floridae; Chordata), and a brachiopod (Lingula

anatina; Brachiopoda). Invertebrate metaxin proteins, like the vertebrate metaxins, have predicted

secondary structures that are dominated by alpha-helical segments, with very little beta-strand.

The pattern of alpha-helical segments is highly conserved between species. Invertebrate metaxin 1

and metaxin 2 proteins each have 9 helices. Eight of the helices are shared by both metaxin

proteins. Metaxin 1 has a C-terminal helix (H10) that metaxin 2 lacks, while metaxin 2 has an N-

terminal helix (H1) that metaxin 1 lacks. The conservation of helices is striking, considering that

the two metaxins only share about 20% sequence identities. For example, the metaxin 1 and 2

proteins of the California sea hare Aplysia californica (Mollusca) have only 21% identities. This

result demonstrates how invertebrate metaxin secondary structure is more highly conserved than

primary structure. (B) shows the alignment of representative insect metaxin 1 and 2 protein

sequences. The alpha-helical segments, labeled H1 to H10, are underlined and in red. The insects

included are the Florida carpenter ant (Camponotus floridanus; Order: Hymenoptera), the

mountain pine beetle (Dendroctonus ponderosae; Coleoptera), the Mediterranean fruit fly

(Ceratitis capitata; Diptera), the drywood termite (Cryptotermes secundus; Blattodea), the

silverleaf whitefly (Bemisia tabaci; Hemiptera), and the common eastern bumblebee (Bombus

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impatiens; Hymenoptera). As in Figure 4(A), the insect metaxins have 9 alpha-helical segments

that are highly conserved between species. Eight of the helices (H2 to H9) are found for both

metaxin 1 and metaxin 2. Metaxin 2 has an extra N-terminal helix (H1), and metaxin 1 has an

extra C-terminal helix (H10).

(A) Human Metaxin 1/C.elegans Metaxin 1

Identities = 31%, Similarities = 46%

Human 1 MAAPMELFCWSGGWGLPSVDLDSLAVLTYARFTGAPLKVHKISNPWQSPSGTLPALRTSH 60 M EL W +GLP++D+ SL L ++ +P++V + + PW+SPSG LP + + C.elegans 1 M----ELHIWPSDFGLPTIDVVSLQFLACSKMCASPVRVIQSTRPWRSPSGELPMVAQTE 56

Human 61 GEVISVP--HKIITHLRK--EKYNADYDLSARQGADTLAFMSLLEEKLLPVLVHTFWIDT 116 GE V K + L+K + D DL+ + A AF L L P ++HTFW D C.elegans 57 GEAKPVTDFEKFVDILKKCGQDVVIDADLTTIEKAQLDAFSCYLHHNLYPAVMHTFWTDE 116

Human 117 KNYVEVTRKWYAEAMPFPLNFFLPGRMQRQYMERLQLLTGEHRPEDEEELEKELYREARE 176 NY VT+ WYA + FP N + ++++ + L+LL G++ + E+ +EA C.elegans 117 LNYNTVTQYWYASHLHFPYNLYY---LEKRRKKALRLLAGKN------DTEILKEAFM 165

Human 177 CLTLLSQRLGSQKFFFGDAPASLDAFVFSYLALLLQAKLPSGKLQVHLRGLHNLCAYCTH 236 L LS +LG KFF G+ P SLDA VF YLA LL+ LP+ +LQV L NL + C.elegans 166 ALNTLSTKLGDNKFFCGNKPTSLDALVFGYLAPLLRVPLPNDRLQVQLSACPNLVRFVET 225

Human 237 ILSLYFP------WDGAEVPPQRQTPAGPETEE------EPYRRRNQILSV 275 + S+Y P W + A TEE E R+ IL C.elegans 226 VSSIYLPLGEDELKRQQANRKMWQSRISKAKADKEAAKTTEEASESLPEEPPMRDAILFT 285

Human 276 LAGLAAMVGYALLSGIVSIQRATPARAPGTRTLGMAEEDEEE 317 L L + +A+ +G++ + EE+ E C.elegans 286 LGALTLSLVFAIHTGLIQVS------VEEEISE 312

(B) Human Metaxin 2/C.elegans Metaxin 2

Identities = 36%, Similarities = 57%

Human 1 MSLVAEAFVSQIAAAEPWPENATLYQQLKGEQILLSDNAASLAVQAFLQMCNLPIKVVCR 60 M+ AA+ W E+ +L+ +Q L+ D A LAVQ FL+M +LP V R C.elegans 1 MN------AAQDW-EDVSLFTPYLNDQALMYDFADCLAVQTFLRMTSLPFNVRQR 48

Human 61 ANAEYMSPSGKVPFIHVGNQVVSELGPIVQFVKAKGHSLSDGLEEVQKAEMKAYMELVNN 120 N +++SP G VP + + +++ IV FV KG +L+ L E Q A+M+A + ++ + C.elegans 49 PNVDFISPDGVVPLLKINKTLITGFNAIVDFVHKKGVTLTSHLSETQVADMRANISMIEH 108

Human 121 MLLTAELYLQWCDEATVGEITHARYGSPYPWPLNHILAYQKQWEVKRKMKAIGWGKKTLD 180 +L T E ++ W + T ++T RYGS Y WPL+ +L + K+ ++ ++ W KT+D C.elegans 109 LLTTVEKFVLWNHDETYDKVTKLRYGSVYHWPLSSVLPFVKRRKILEELSDKDWDTKTMD 168

Human 181 QVLEDVDQCCQALSQRLGTQPYFFNKQPTELDALVFGHLYTILTTQLTNDELSEKVKNYS 240 +V E D+ +ALS +LG+Q Y PTE DAL+FGH+YT++T +L ++ +K YS C.elegans 169 EVGEQADKVFRALSAQLGSQKYLTGDLPTEADALLFGHMYTLITVRLPLTNITNILKKYS 228

Human 241 NLLAFCRRIEQHYFEDRGKGRLS 263 NL+ F +RIEQ YF+ C.elegans 229 NLIEFTKRIEQQYFKQ 244

Figure 1(A,B). Homology of invertebrate metaxins: amino acid sequence alignments.

Identities = 27%, Similarities = 45%

Human 1 MAAPME--LFCWSGGWGLPSVDLDSLAVLTYARFTGAPLKVHKISNPWQSPSGTLPALRT 58 M + L+ + G +GLPS+D + L L RFT P+ V SNP +S +G LP L+ D.melanogaster 1 MEMQLGAMLYVYKGEYGLPSIDFECLRALCLLRFTRCPMDVQTSSNPLRSGAGKLPYLQI 60

Human 59 SHGEVISVPHKIITHLRKEKYNADYDLSARQGADTLAFMSLLEEKLLPVLVHTFWIDTKN 118 + + +I L E Y D LS +Q + A+ + + L + + + N D.melanogaster 61 GNQKFAGY-RQIKRVLDLEGYPIDAHLSTKQKHLSTAYANWVFTNLHAYYHYFLFGEPHN 119

Human 119 YVEVTRKWYAEAMPFPLNFFLPGRMQRQYMERLQLLTGEHRPEDEEELEKELYREARECL 178 + TR YA+ PFP NF+ P QR+ + +Q++ G + ++ E + D.melanogaster 120 FDTTTRGLYAKRTPFPFNFYYPSSYQREACDVVQVMAGFDVNDKLDKHEGDY------171

Human 179 TLLSQRLGSQKFFFGDAPASLDAFVFSYLALLLQAKLPSGKLQVHLRGLHNLCAYCTHIL 238 S++LG + +FFGD + DA V+SYLA++ + LP+ LQ H++G NL + I D.melanogaster 172 ---SRKLGRKVWFFGDTYSEFDAIVYSYLAIIFKIALPNNPLQNHIKGCQNLVNFINRIT 228

Human 239 SLYFPWDGAEVPPQRQTPAGPETE----EEPYRRRNQILSVLAGLAAMVGYALLSGIVSI 294 F +G +TP+G E E + ++AG+ A++ + I D.melanogaster 229 KDIFRIEGYSSVKLTKTPSGTEASLTASERKFLDSELNTKIVAGVGAVLAMGAFAAWRGI 288

Human 295 QRATPARAPGTRTLGMAEEDEE-----E 317 R+ T G+ ED+E + D.melanogaster 289 YNQVTTRS-STDYDGIDYEDDEMEEGLD 315

(D) Human Metaxin 2/Drosophila melanogaster Metaxin 2a (CG8004)

Identities = 41%, Similarities = 61%

Human 1 MS--LVAEAFVSQIAAAEPWPENATLYQQLKGEQILLSDNAASLAVQAFLQMCNLPIKVV 58 M+ +++ + +AEPWPE+ATLYQ + EQILL +NA+ LAV+A+L+MCNLP + D.melanogaster 1 MTSQYLSQLITADKLSAEPWPEDATLYQPYEAEQILLPENASCLAVKAYLKMCNLPFLIR 60

Human 59 CRANAEYMSPSG---KVPFIHVGNQVVSELGPIVQFVKAKGHSLSDGLEEVQKAEMKAYM 115 ANAE+MSP G K+PFI G + +E PIV FV+ K ++ +E +KA+M+ Y+ D.melanogaster 61 SCANAEHMSPGGRMTKLPFIRAGAFIFAEFEPIVNFVEQKELAIGSWQDEDEKADMRTYV 120

Human 116 ELVNNMLLTAELYLQWCDEATVGEITHARYGSPYPWPLNHILAYQKQWEVKRKMKAIGWG 175 LV N+ AELY+ + +E E+T R G +PWPLNH+ Y K+ R +K W D.melanogaster 121 SLVENIFTMAELYISFKNERVYKEVTAPRNGVVFPWPLNHMQNYGKRRNALRLLKVYQWD 180

Human 176 KKTLDQVLEDVDQCCQALSQRLGTQP---YFFNKQPTELDALVFGHLYTILTTQLTNDEL 232 +D V++ V +CC+ L +L P +F+ QP ELDA+ FGHL++ILTT L N L D.melanogaster 181 DLDIDSVIDKVAKCCETLEYKLKESPETPFFYGDQPCELDAIAFGHLFSILTTTLPNMAL 240

Human 233 SEKVKNYSNLLAFCRRIEQHYFEDRGKGRLS 263 +++ YF+ R + D.melanogaster 241 AQTVQKFQHLVEFCRFVDEKYFQTRCLP--N 269

Figure 1(C,D). Homology of invertebrate metaxins: amino acid sequence alignments.

(A) Invertebrate Metaxins 1 and 2 Protein Domains

Ciona intestinalis (sea squirt, vase tunicate) metaxin 1

(1-314)

Nematostella vectensis (starlet sea anemone) metaxin 1

(1-325)

Trichoplax adhaerens metaxin 1

(1-292)

Ciona intestinalis (sea squirt, vase tunicate) metaxin 2

(1-261)

Nematostella vectensis (starlet sea anemone) metaxin 2

(1-244)

Trichoplax adhaerens metaxin 2

(1-231)

GST_N_Metaxin1_like; GST_C_Metaxin1_3; Tom37

GST_N_Metaxin2; GST_C_Metaxin2

Figure 2(A). Domain structure of invertebrate metaxin proteins. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.895979; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

(B) Insect Metaxins 1 and 2 Protein Domains

Aedes aegypti (yellow fever mosquito) metaxin 1

(1-334)

Zootermopsis nevadensis (Nevada dampwood termite) metaxin 1

(1-321)

Cimex lectularius (common bed bug) metaxin 1

(1-318)

Aedes aegypti (yellow fever mosquito) metaxin 2

(1-265)

Zootermopsis nevadensis (Nevada dampwood termite) metaxin 2

(1-262)

Cimex lectularius (common bed bug) metaxin 2

(1-317)

GST_N_Metaxin1_like; GST_C_Metaxin1_3; Tom37

GST_N_Metaxin2; GST_C_Metaxin2

Figure 2(B). Domain structure of invertebrate metaxin proteins. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.895979; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

(A) Invertebrate and Vertebrate Metaxins

Not Including Insects

Crown-of-thorns starfish metaxin 1

Purple sea urchin metaxin 1

Pacific oyster metaxin 1

Snail (Biomphalaria glabrata) metaxin 1

Atlantic horseshoe crab metaxin 1

// Bark scorpion metaxin 1

Human metaxin 1

Mouse (Mus musculus) metaxin 1

Xenopus laevis metaxin 1

Zebrafish (Danio rerio) metaxin 1

Crown-of-thorns starfish metaxin 2

Purple sea urchin metaxin 2

Snail (Biomphalaria glabrata) metaxin 2

Pacific oyster metaxin 2

Atlantic horseshoe crab metaxin 2 // Bark scorpion metaxin 2

Zebrafish (Danio rerio) metaxin 2

Xenopus laevis metaxin 2

Human metaxin 2

Mouse (Mus musculus) metaxin 2

Figure 3(A). Evolutionary relationships of metaxin 1 and 2 proteins of invertebrates. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.895979; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

(B) Insect Metaxins

Amyelois moth metaxin 2

Domestic silkworm metaxin 2

Western honey bee metaxin 2

Common eastern bumblebee metaxin 2

// Drosophila elegans metaxin 2a

Drosophila eugracilis metaxin 2a

Drosophila melanogaster metaxin 2a (CG 8004)

Drosophila eugracilis metaxin 2b

Drosophila elegans metaxin 2b

Drosophila melanogaster metaxin 2b (CG5662)

Drosophila elegans metaxin 1

Drosophila eugracilis metaxin 1

Drosophila melanogaster metaxin 1 (CG9393)

// Western honey bee metaxin 1

Common eastern bumblebee metaxin 1

Amyelois moth metaxin 1

Domestic silkworm metaxin 1

Figure 3(B). Evolutionary relationships of metaxin 1 and 2 proteins of invertebrates. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.06.895979; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

(A) Invertebrate Metaxin 1 and 2 Amino Acid Sequences

H1 H2

1 MAATV ------MELQS-WPGD--WNLPSIDTRCLEVLAYAKF AGCPINVN-RNNNPWKSPLGEFPVLRSGG 61 Coral MTX 1

1 MASFG[ 1]GSEEIRSKMTLYV-FTGN--EYSASVDPESLQILTYLRL[6]ENEEIEIK-YTCKHWESPTGVFPTLVSDG 76 Sponge MTX 1

1 ------MELHV-WEGDssYGLPSMDLESLQIMMYVKF SGAPVKII-KSMSPAHAPSRNLPVLSCPS 58 Amphipod MTX 1

1 MSSL-[10]ASVPWPDDAALYQPYEKE--QILLTDSANCLSVKTFLKM CGLNFNLELRVNAEEMSPSGKIPFIKVGP 80 Octopus MTX 2

1 ----- ASEPWPDDVCLYQPYKLQ--QVLLPDNAQCLAVQCYLQM CGLPFRTVLRTNAEHMSPSGKPPFLRAGP 66 Amphioxus MTX 2

1 MTTLK[12]AKEPWPDNVNLYQTYEAG--QILLPDSASCLSVKAFLEM CGLKYSVVLRNNAEHMSPSGKVPFIQIGP 83 Brachiopod MTX 2

H3 H4 H5

62 DVLTAPDKIMDYFRLKNFNADYQLNAKQQADTLALVSLVRSKLYPAISFTFWVDERNYTKFTR--PWFSKRLPFPLNYFL 139 Coral MTX 1

77 LYYTGVDSIIKELKKQGLDLDADCSDEELAEISAYSAFLSDRLKKALLYLHWVEAKNYTEVTRgwLSGATVFPFTLYILS 156 Sponge MTX 1

59 GNLNKFSAITAHLRRKNFSCDHELTRKQCSDVLAYQQLIEEKLFPAFVYLWWVEPRSYQEIIH--KWYYSRMRFPYKLWV 136 Amphipod MTX 1

81 FLISEMDPIIAFVNTKGFHISNHLSDSQQSEMRAYMALVESILINAELYLSWCHEDVYYEITK--PRYGACYPWPLNMIL 158 Octopus MTX 2

67 YVISEVDPIIKFVSTKGHSLSDKLDRPQQAEMKAYLALVQGTLGNAELYISWCDPTTASEISR--PRYSSPYPWPLNTLL 144 Amphioxus MTX 2

84 FLISEFEPIIGFVNTKGFHLSANLDDTERAEMRAYMALVDNILVNAEHYISWCDATVLQTVTK--PRYGSPYPWPLNKLV 161 Brachiopod MTX 2

H6 H7 H8

140 PGKlSRQKRSEICEGVYSHEIDDLQLEN---KLFKEALECLKLLSIVLGDKDFFFSHRPTTLDAVVFSHLAVIYKAPLP- 215 Coral MTX 1

157 RQH-RLAKQVIMTSSSGNTSNNTSTIEQcgiELLSQAEEAISLLAGRLGDQKYFYGSSLSSFDVLVFGYLDLIAQYELPg 235 Sponge MTX 1

137 PSN-RHNAYKAFIESIVDEPDDEVAVET---ELLKRGQECLTLLSVRLGDKGFFFGSSPSSLDAILAPYLALLLKVDLR- 211 Amphipod MTX 1

159 PLK----KFTEIKNRLTSIGWFNKSFE----EVCEQIKTCCQVLSERLGNQTYFFGDKPTELDALVFGHLFTLLTTNLP- 229 Octopus MTX 2

145 AYR----KQWDVQNQLKALGWYSKTLE----EVYDEVDNCCKALAQRLGNHHYFFKQGPTELDALVFGHLYTILTTPLT- 215 Amphioxus MTX 2

162 PLK----KWYEETSRLKALGWSQKTLS----EVCEEVQTCCQALSERLDKNNFFFGNRPTELDALVFGHLFTILTTPMP- 232 Brachiopod MTX 2

H9 H10

216 NNKLHNYLQGYDNLSTFCRRVLQRYFS[6]QRQQDQPSAKRSSSQDDAAVDRNS[17]TAYAFISGLV[12]EESTPELG 319 Coral MTX 1

236 NNQLGTRLKSLVQLTNFCSDIREKAYP EIKSRHVSTSSSMSPSSPLRKDPT[ 1]WIFVGVAGIL[ 3]QGMRVGLT 308 Sponge MTX 1

212 VPALQNHLRQCPNLTAYVSRALATCFP NENPSSIGYGRDKPRGPPPAEDGV[16]FGYVFASGLV[ 9]DGEEYGLA 305 Amphipod MTX 1

230 VDQFALVIQKYDNLVEFCNNIQATYYK ------256 Octopus MTX 2

216 DNSLADIVKEYSNLINFCRHIEKKFFE ERERGPKD------250 Amphioxus MTX 2

233 VNKLAEIVKNFDNLVHFCARIEQQYFS ATEAS------264 Brachiopod MTX 2

320 YTFEG[10] 334 Coral MTX 1

309 RKLLK 313 Sponge MTX 1

306 EEEEE[ 4] 314 Amphipod MTX 1 Figure 4(A). Secondary structures of invertebrate metaxin proteins: multiple sequence alignments.

(B) Insect Metaxin 1 and 2 Amino Acid Sequences

H1 H2

1 ------MDNAEI------LQLDI-WKGDWGLPSVDIDCLQVLAYAKFSGIPLKVNLTSN-PFKTPNGRLPLLRA 60 Ant MTX 1

1 ------MLGNDK-----FTLYV-YDGDYGLPSFNVECTKVLLYIAMVKVSVEIKTLNN-IKSCAFYTGPCFMH 60 Beetle MTX 1

1 ------MLLGG------VLYV-YKGEWGLPSIDFDCLRALCLVKFTRCPLQIDTNVN-PMRSSTGKLPYIQI 58 Fruit fly MTX 1

1 -MPSVLLTESIALELGAQEPWPQDVKLFQPYEVEQILLPDNANCLAVQAFLKMCGLDFEVEPRSNAEYMSPSGRVPFIKC 79 Termite MTX 2

1 mVSSNVLLDSIQIEIGGDNEWPNDAKLFQPYETEQILMPDSANCLAVQAFLQMANLKYETVYKANAAEMSPTGRVPFIKV 80 Whitefly MTX 2

1 -MPCPLLADSITLELEAQEPWPQPITLYQPYEVEQILLPDNANCLAVQAFLKMCGLDFQIEPRSNAEYMSPSGRVPFIKC 79 Bumblebee MTX 2

H3 H4 H5

61 GLNTLDTVKDILPFFRAKHYNSDYTLTDKQCADVLAYDALLKEKLYPALQFIWWIDKKNLD____ELIRPWYCKALPFPF 136 Ant MTX 1

61 KNLSFGSFGDIVPYLNTLNFDLDVQLSPKQRSEALALSNMVQAKLRGPVEFAFWVDYRNFE[41]ELVK------166 Beetle MTX 1

59 GTERFCGYREIKKMLDKEGFAIDAKLQVKHELLSPAYANWVFTNLHSYYNYFMYGEPNNFD____-TTRSLYAKRTMFPL 133 Fruit fly MTX 1

80 GAFVVAELDPIISFVNHKGISLSDKLDSVQKADMRAYMSLINTVLANAELYITWCDKTTLQ____EVTKPRFGSVLPWPL 155 Termite MTX 2

81 GQYLVAELEPIVAFVQNKNISLTSNLTDAEKSDMRAYMSLINTVFGNAENYITWCDPETYS____KVTKKRFGSIHPFPL 156 Whitefly MTX 2

80 GAFLISEFDNIVSFIGNKGRSLSDHLSATCKADMRAYMSLVNNVFVNAELYICWVDEPTLN____EVTKVRHGSVYPWPL 155 Bumblebee MTX 2

H6 H7 H8

137 KFYYPGKFERQAQALMQSLYSME DNID[4]EVYSEAQKCLTLLSMRLGDGDFFFGQQ-PSTIDAIVYSYLALLLKAP 213 Ant MTX 1

167 ------EYLN ---SLASECLASLSARLGVSKYFFRDQ-PSSLDVVVYSYVAPLVKLP 213 Beetle MTX 1

134 NFFYPSSYQREACDIVMVMSGFD[5]ENHD[2]SLTINAKKCVNLLSKKLGKKVWFFGDV-FSEFDAIVYSYLAILFKTT 213 Fruit fly MTX 1

156 NHILAWQKRSQVVKKLGVLGWGN KTLD EVYEEIENCCSALSERLENRPYFFGDR-PTELDAQVFGHLFTILTTP 228 Termite MTX 2

157 NHILVWKKRSATIERMKALGGKT EQ-E___AVLDDVDLCCQSLSDRLGDREFFFKSG-PTELDALVFGHIFTIITTP 228 Whitefly MTX2

156 NHFLNWQKRKEVIKKLNVLGWYN KTIE___EVCKEVQNCCTALSERLEGSDYFSGEKtPNELDALVFGHIFTIVTTP 229 Bumblebee MTX 2

H9 H10

214 LPNPVLQNHLRNCTNLVKYVSRISQRYFEYE[4]EKDKAEKNAEKLgeDECEFPNKRRNQIFAAFVAAMAmaGYALSTGI 294 Ant MTX 1

214 FPSNDLSIVVALWPNLVSFINRIDAKFLPEL[1]KESKYIKNEAKLktSDDEVSYVAISILTVSAISLLW--GFAVSKGF 289 Beetle MTX 1

214 LPNNPLQNHIKGCQNLVNFINRITKDIFKNE[2]NSVKAIKSQSSN--DPLLTATERHFLESEKKTKILAgiGAVVAMGT 290 Fruit fly MTX 1

229 LPNNRFASIVRGYPNLIELCKLVEKEYF------ERTAEA------262 Termite MTX 2

229 LPNNQLNGIVRGFPNLIEHCKRIDKMFF------KTNFSSEWM------265 Whitefly MTX 2

230 LPGNKLANIVQSYPLLVHLCKRIETSIF------SPQEIRVK------265 Bumblebee MTX 2

295 V[12]STEYI[7] 319 Ant MTX 1

290 I SSKLF 295 Beetle MTX 1

291 F[12]SKRYY[3] 311 Fruit fly MTX 1

Figure 4(B). Secondary structures of invertebrate metaxin proteins: multiple sequence alignments.