Discovering the ancient protein family that shapes cellular machinery and human health
Within every cell in our bodies, a microscopic universe of complex machinery operates with breathtaking precision. Among the most mysterious components of this cellular factory are membrane proteins—sophisticated molecular devices that control what enters and exits our cells' specialized compartments.
For years, scientists had noticed that two particular proteins, TMEM41B and VMP1, played crucial roles in cellular processes like autophagosome formation (the creation of cellular "recycling centers"), lipid droplet homeostasis, and lipoprotein secretion.
Both were known to be essential for health—mutations in these proteins could disrupt cellular recycling systems and contribute to disease—but their evolutionary origins and molecular functions remained deeply puzzling.
When researchers Fumiya Okawa, Yutaro Hama, and Sidi Zhang began collaborating in Noboru Mizushima's lab at the University of Tokyo, they recognized something intriguing: both TMEM41B and VMP1 contained a similar structural region that resembled domains found in bacterial proteins called DedA family proteins. This observation sparked a compelling scientific detective story that would take them deep into the evolutionary history of cellular machinery—a journey that would ultimately reveal an entire superfamily of proteins with surprising connections to human health and disease 1 2 .
Proteins rarely invent entirely new structures from scratch. Instead, evolution tinkers with existing designs, modifying and repurposing them for new functions. Okawa, Hama, and Zhang suspected that TMEM41B, VMP1, and bacterial DedA proteins might share a common ancestor—an ancient protein that existed before the divergence of bacteria and eukaryotes billions of years ago.
To test this hypothesis, the team employed sophisticated computational biology techniques to search for remotely related proteins across hundreds of species. Their investigation revealed not just three related protein families, but four distinct groups that together formed what they termed the DedA superfamily 1 :
| Family Name | Key Members | Primary Organisms | Cellular Functions |
|---|---|---|---|
| TMEM41 | TMEM41B | Eukaryotes | Autophagosome formation, lipid homeostasis |
| VMP1 | VMP1 | Eukaryotes | Autophagosome formation, lipoprotein secretion |
| DedA | Various DedA proteins | Bacteria | Membrane integrity, alkaline tolerance |
| PF06695 | Previously uncharacterized proteins | Mainly bacteria | Unknown (likely membrane-related) |
What made this discovery particularly significant was the distribution of these proteins across life's domains. Since all four families included members from bacteria and archaea, the researchers concluded that the DedA superfamily must have originated very early in evolutionary history—before the last universal common ancestor of all extant life 1 6 .
Unraveling evolutionary relationships across billions of years requires specialized tools. Traditional phylogenetic methods often struggle when protein sequences become too diverged to align properly. To overcome this challenge, Okawa, Hama, and Zhang utilized an innovative approach called graph splitting 7 .
This computational technique represents proteins as interconnected nodes in a vast network based on sequence similarity. By strategically dividing this network, researchers can reconstruct evolutionary relationships even when sequences have diverged beyond recognition by conventional alignment methods.
The graph splitting approach proved particularly valuable for analyzing the DedA superfamily, whose members had evolved along distinct paths for millennia yet retained structural hints of their common ancestry 6 7 .
A computational method that maps evolutionary relationships through sequence similarity networks
With the evolutionary relationships established, the team turned to perhaps their most intriguing question: what molecular structure might these proteins share? Using coevolution-based structural prediction methods, they analyzed patterns of amino acid changes across the DedA superfamily.
Their computational models suggested something surprising: the shared DedA domain likely contained two reentrant loops—unusual structural features where a protein helix dips partway into the membrane before looping back out on the same side.
To test their prediction, the researchers turned to biochemistry. They employed the substituted cysteine accessibility method (SCAM), a technique that systematically replaces amino acids with cysteine residues and then tests their accessibility to chemical modification.
Their biochemical results confirmed the computational predictions: the DedA domain indeed contained two reentrant loops facing each other in the membrane.
| Technique | Application in This Study | Key Insight Gained |
|---|---|---|
| Remote homology search | Identified distantly related proteins | Revealed the full extent of the DedA superfamily |
| Graph splitting phylogenetics | Reconstructed evolutionary relationships | Established common ancestry of four protein families |
| Coevolution-based structural prediction | Predicted 3D structure from sequence data | Identified two reentrant loops in the DedA domain |
| Substituted cysteine accessibility method (SCAM) | Mapped membrane topology | Biochemically validated predicted reentrant loops |
Cutting-edge science requires sophisticated tools. The investigation into the DedA superfamily employed a diverse array of research reagents and techniques, each providing crucial pieces of the puzzle.
| Reagent/Technique | Function/Application | Role in This Study |
|---|---|---|
| SCAM (Substituted Cysteine Accessibility Method) | Mapping membrane protein topology | Validated predicted reentrant loops in DedA domain |
| HaloTag Technology | Protein labeling and purification | Enabled pulse-chase assays for protein turnover studies 5 |
| CRISPR-Cas9 Screening | Genome-wide gene knockout | Identified TMEM41B as essential for autophagosome formation 4 |
| Graph Splitting Algorithms | Phylogenetic reconstruction of distantly related proteins | Established evolutionary relationships within superfamily 7 |
| Coevolution Analysis | Predicting protein structure from sequence data | Predicted reentrant loop structure of DedA domain |
| Lipid Probes | Detecting membrane lipid composition | Studied lipid scrambling activity of TMEM41B and VMP1 |
While TMEM41B and VMP1 were initially studied for their roles in autophagy, the discovery of their membership in the DedA superfamily suggested much broader functions.
Subsequent research confirmed that these proteins act as phospholipid scramblases—enzymes that shuttle lipids between the two layers of cellular membranes .
This scrambling activity is crucial for expanding autophagosome membranes during their formation. Without properly functioning TMEM41B or VMP1, cells cannot create these essential recycling compartments, leading to accumulation of damaged components and cellular dysfunction.
Beyond autophagy, these proteins are now known to be essential for lipoprotein secretion and lipid droplet homeostasis—processes fundamental to cellular metabolism 1 6 .
In bacteria, DedA family proteins have been linked to antimicrobial resistance, particularly to colistin .
Proper TMEM41B function appears essential for viral replication—some viruses hijack this protein to create replication membranes.
VMP1 dysfunction has been linked to pancreatic cancer and metabolic disorders 1 .
The collaborative work of Fumiya Okawa, Yutaro Hama, and Sidi Zhang represents a compelling example of how evolutionary insights can illuminate modern cellular biology. By recognizing the ancient family relationship between seemingly unrelated proteins across life's domains, they uncovered fundamental principles of cellular organization.
Their research journey—from computational predictions of protein structure to biochemical validation—exemplifies the interdisciplinary nature of modern biology. The discovery that TMEM41B and VMP1 belong to an ancient superfamily with potential ion-coupled transport functions provides a new framework for understanding how cells manage their internal membranes and control the movement of substances between compartments.
What began as a curiosity about similar sequences in proteins across evolutionary time has grown into a rich understanding of fundamental cellular processes—demonstrating that sometimes, to understand how our bodies work today, we must first understand the deep evolutionary history written in our genes.