The Hidden World of Membrane Microproteins

Abstract

Proteins are critical components of biological membranes and play key roles in many essential cellular processes. Membrane proteins are a structurally and functionally diverse family of proteins that have recently expanded to include a number of newly discovered tiny proteins called microproteins, or micropeptides. These microproteins are generated from small open reading frames which produce protein products that are less than 100 amino acids in length. While not all microproteins are membrane proteins, this review will focus specifically on this subclass to highlight some of the important biological activities that have been ascribed to these molecules and to emphasize their promise as exciting new players in membrane biology.

1. Introduction

Biological membranes are dynamic structures that provide specialized permeability barriers for cells and subcellular organelles and are essential for life. Both prokaryotic and eukaryotic cells have a plasma membrane that forms an outer boundary of each cell and separates the interior of the cell from the external environment. Unlike prokaryotes, eukaryotic cells also contain internal membrane structures that define discrete organelles that perform specialized functions mediated by their unique microenvironments. Membranes are lipid bilayer structures that are primarily comprised of phospholipids, cholesterol and proteins held together by non-covalent forces. Membrane proteins account for roughly half the mass of most cellular membranes and they mediate critical cellular processes including ion transport, signal transduction, respiration, motility and cell-cell communication [1]. It has been estimated that one third of all protein-coding genes encode membrane proteins, and the importance of these proteins is highlighted by the fact that nearly half of all current therapeutic targets are membrane proteins [2, 3].

Recently, the proteome has expanded to include a novel class of small proteins called microproteins, or micropeptides. These microproteins are translated from small open reading frames (smORFs or sORFs) of less than 300 nucleotides in length to generate proteins that are 100 amino acids or smaller [4]. Due to their small size, many microprotein-coding genes have been unintentionally overlooked by standard gene annotation methods and have been incorrectly classified as noncoding RNAs. In recent years, a concentrated effort has been made to identify protein-coding smORFs, and innovative bioinformatic and technological advances have led to the discovery of hundreds of putative microproteins [5–11]. Interestingly, a high proportion of these microproteins are predicted to contain transmembrane α-helix motifs (Figure 1), suggesting that microproteins may represent a rich source of uncharacterized membrane proteins [12, 13]. To date, only a limited number of microproteins have been functionally characterized, and these proteins have been shown to play roles in a broad range of critical cellular functions including development, differentiation, stress signaling and metabolism (Figure 2)[14, 15]. The focus of this review will be to highlight the important roles that have been ascribed to membrane microproteins and to discuss the exciting potential these proteins hold as novel players in membrane biology.

Figure 1. Membrane microproteins exert their biological effects through protein-protein interactions.

A large proportion of putative microproteins are predicted to contain hydrophobic domains that target them to cellular membranes. Within these membranes, microproteins function as singular protein domains that engage much larger regulatory proteins or multiprotein complexes to exert powerful biological functions. Shown here is a ribbon model of the structure of the microprotein phospholamban (blue) bound to the calcium pump SERCA (orange) (PDB ID 4KYT) [45]. This image highlights how microproteins can fit into small binding clefts in large regulatory proteins to fine-tune very complex biological systems. This figure was generated using Mol* at RCSB PDB [46, 47].

Figure 2. Functions of membrane microproteins.

Microproteins have been shown to localize to the plasma membrane and to the membranes of intracellular organelles where they play essential roles in regulating critical biological processes. The numbers in this figure correspond with the different protein-microprotein complexes detailed in the text and illustrate the many different functions that have been ascribed to membrane microproteins (indicated in blue). The protein complexes and biological processes they regulate are designated by the following numbers: 1-Endoplasmic reticulum (ER) membrane microproteins (PLN, SLN, MLN, ALN, ELN or DWORF) and their regulation of SERCA-mediated calcium transport; 2-The modulation of the Na⁺,K⁺-ATPase at the plasma membrane by FXYD microproteins; 3-The regulation of mitochondrial enzyme complexes by mitoregulin/MOXI; 4-The initiation of stress signaling through PIGBOS-mediated inter-organelle communication; 5- The association of SPAR with the v-ATPase to recruit proteins that inhibit mTORC1 activation; 6-The fusogenic role of the plasma membrane skeletal muscle microprotein myomerger/ myomixer/minion.

2. Functions of Membrane Microproteins

Membrane proteins play essential roles in coordinating and executing the movement of materials and information across cell membranes. While gases and small hydrophobic molecules can diffuse directly across the phospholipid bilayer, membrane proteins are required for the transport of molecules that are too large (sugars, amino acids) or charged (ions) to cross the membrane. Membrane proteins also play critical roles in cell-cell communication as they serve as receptors for ligands such as neurotransmitters, hormones and growth factors, which initiate downstream signaling events. Additionally, membrane proteins are required for physical cell-cell interactions and regulate cell adhesion, cellular coupling and membrane fusion. The membrane proteins that mediate these various processes are often very large in size, with examples including ion channels, transporters, exchangers, receptors and enzymes (Figure 2). While these families of proteins have been extensively studied, many aspects of their biology, including their regulation, assembly and localization, are not yet fully understood. With the recent emergence of microproteins as an untapped reservoir of putative membrane molecules, it is exciting to speculate that these tiny proteins could be some of the critical missing pieces to the puzzle of membrane protein biology. In the following sections, we will introduce a number of recently described membrane microproteins and review their biological functions to highlight their fundamental importance as essential membrane proteins.

2.1. Membrane Microproteins Regulate Ion Transport

A major function of the plasma membrane and the membranes of intracellular organelles is to maintain steady-state asymmetric concentrations of cations and anions, and the regulation of these gradients is critical for a host of fundamental cellular functions including energy production, intracellular signaling, membrane depolarization and muscle contraction [16]. Specialized ion transport proteins and multicomponent complexes are required to coordinate ion flux, and many of these transporters are regulated by small auxiliary proteins. Several membrane microproteins have been shown to function as regulators of ion transporters, and it has been hypothesized that their small size enables them to fit into larger protein complexes to regulate their activity [15](Figure1).

Among the best characterized examples of membrane microproteins is a family of calcium regulatory proteins that bind to the sarco/endoplasmic reticulum (S/ER) calcium ATPase (SERCA) in the S/ER membrane and modify its activity by altering its affinity for calcium (Figure 2.1)[17, 18]. These include the well-known SERCA inhibitors phospholamban (PLN)[19] and sarcolipin (SLN)[20], as well as the more recently identified inhibitory proteins myoregulin (MLN)[21], endoregulin (ELN)[22] and another-regulin (ALN)[22] and the SERCA activating microprotein DWORF [23]. The identification of the homologous SERCA inhibiting protein sarcolamban (SCL) in Drosophila [24] highlights the evolutionary conservation of this system. SERCA activity is essential for maintaining calcium homeostasis in all cell-types and tissues and it has been proposed that these various SERCA regulatory peptides may contribute to the specialized calcium-handling properties of different cell-types, as they each have unique tissue expression profiles [18, 22]. PLN (ventricle), SLN (atria) and DWORF are expressed in the heart, while MLN is restricted to skeletal muscle and ELN is epithelial and endothelial cell-specific, while ALN is ubiquitously expressed in all cell-types and tissues [22]. Although the inhibitory peptides PLN, SLN, MLN, ELN, ALN and SCL have few conserved residues, they all adopt a similar conformation with conserved amino acids positioned in the same orientation and these residues form a hydrophobic binding motif for SERCA [17, 21, 22, 24].

While SERCA may be the most extensively studied example, many other P-type ATPases are also regulated by membrane microproteins [25–28]. The sodium-potassium (Na⁺,K⁺)-ATPase is regulated by a family of membrane microproteins known as the FXYD family, which is comprised of seven different proteins in mammals and includes the widely studied muscle-enriched protein phospholemman (FXYD1)[26, 28]. The major function of the FXYD proteins is regulation of the Na⁺,K⁺-ATPase in excitable and osmoregulatory tissues (Figure 2.2), but they have also been shown to modulate the activity of other transporters including the Na⁺,Ca²⁺ exchanger, the L-type Ca²⁺ channel and the gastric-type H⁺,K⁺-ATPase [29]. Similar to the SERCA-regulating peptides, the FXYD family members are tissue-specific, and some members are also developmentally regulated, which allows for more finely tuned regulation of the pump [25, 27]. FXYD1 (phospholemman) is expressed in heart and skeletal muscle, FXYD2 (the γ-subunit of the Na⁺,K⁺-ATPase) and FXYD4 (CHIF) are expressed in kidney, FXYD7 is found in the brain, and FXYD3 (MAT-8) is mainly expressed in the stomach, colon and uterus [30]. There are also known examples of P-type ATPases being regulated by microproteins in bacteria with the E. coli inner membrane microprotein KdpF acting as a stabilizing factor for the Kdp P-ATPase complex through a ‘lipid-like’ stabilization [25].

2.2. Membrane Microproteins Play Diverse Roles in Cellular Organelles

As discussed above, a high proportion of microproteins contain hydrophobic domains and therefore favor interactions with membrane bilayers both at the plasma membrane and within subcellular organelles [12]. Several functionally relevant membrane microproteins have been described in the mitochondria [31–33] and recent bioinformatic analysis has predicted that many additional putative microproteins have strong profiles of mitochondrial localization, suggesting that the mitochondria could be a potential “evolutionary playground for a subset of recently evolved small proteins” [34]. Two independent studies identified a novel muscle-enriched mitochondrial membrane microprotein named mitoregulin [33], or MOXI [32], and showed that it localizes to the inner mitochondrial membrane where it interacts with several protein complexes and regulates mitochondrial membrane potential, respiration, calcium retention capacity and fatty acid oxidation (Figure 2.3)[32, 33]. Additionally, several mitochondrial membrane microproteins have also been shown to play critical roles as accessory subunits in the mitochondrial oxidative phosphorylation complex and they assist in the regulation, assembly and stabilization of the core central subunits (Figure 2.3)[35]. An exciting recent study has identified a previously uncharacterized mitochondrial microprotein, called PIGBOS, and shown that it localizes to the outer mitochondrial membrane at endoplasmic reticulum (ER) contact sites and contributes to the signaling events that drive the unfolded protein response (UPR) [31](Figure 2.4). PIGBOS is widely expressed in many different cell-types and tissues, which suggests it is a ubiquitous component of the cellular stress response. The discovery of PIGBOS adds to the growing evidence that membrane associated microproteins may participate in inter-organelle communication to regulate cellular homeostasis or act as stress signaling molecules [31, 36].

Membrane microproteins have also been identified in intracellular sorting organelles, which provides additional evidence for their roles in cellular signaling events. Hemotin was identified in Drosophila to be expressed in hemocytes (Drosophila macrophages) where it localizes to endosomes and plays a key role in regulating endosomal maturation during phagocytosis [37]. The vertebrate homolog of hemotin has been identified as Stannin, which is important for the regulation of organometallic cytotoxicity [38], showing that this regulatory mechanism is also conserved across evolution [39].

2.3. Membrane Microproteins are Involved in Protein Recruitment and Membrane Dynamics

Another important role that has been ascribed to membrane microproteins is the recruitment of proteins to membrane domains to help coordinate signaling events or modulate membrane dynamics. A recently described example of this is SPAR, which has been shown to localize to late endosomes and lysosomes where it interacts with the lysosomal v-ATPase complex [40](Figure 2.5). Importantly, SPAR does not modulate the proton pump activity of v-ATPase through its physical interaction, rather it promotes the association of v-ATPase with the Ragulator-Rag supercomplex, which inhibits the recruitment of mTORC1 to the lysosome and prevents its activation [40, 41]. SPAR is highly expressed in a subset of tissues, including skeletal muscle, and it has been shown that SPAR is downregulated in skeletal muscle in response to injury, which promotes mTORC1 activation and drives muscle regeneration [40]. The cell-type specific expression pattern of SPAR may provide an important mechanism by which mTORClactivation can be regulated in a tissue-specific manner to control the process of muscle regeneration [40].

Membrane microproteins also act at the plasma membrane to drive membrane fusion events, and a quintessential example of this is the skeletal muscle fusion factor myomerger/myomixer/minion, which was recently discovered by three independent groups [42–44]. Myomerger expression coincides with myoblast differentiation and genetic deletion studies have conclusively shown it is required for myoblast fusion and skeletal muscle formation during embryogenesis. The exact mechanism by which myomerger works is not yet known, but current data suggests it is involved in coordinating protein recruitment to the membrane and that it participates in protein-protein interactions to initiate cytoskeletal reorganization events that are critical for fusion [42–44](Figure 2.6). The dramatic phenotype observed in myomerger null mice highlights how critical the activity of membrane microproteins can be and suggests that this growing class of proteins could contain additional uncharacterized members that participate in essential processes that are absolutely required for life.

3. Concluding Remarks

The growing number of recently described microproteins derived from previously unannotated smORFs has increased the complexity and breadth of the cellular proteome. Computational and experimental studies have generated data sets containing hundreds of putative novel microproteins that are awaiting validation and characterization, and these proteins could shed light on many critical unanswered biological questions. Interestingly, there is a high prevalence for predicted α-helical transmembrane domains in these proteins, which are coded for by hydrophobic stretches of amino acids, suggesting that many of these microproteins are targeted to biological membranes [12, 13]. It can be speculated that the localization of these small proteins to cellular membranes may enhance their stability and protect them from the rapid degradation that they may be otherwise susceptible to as small molecules. A common feature of many functional microproteins is that they exert their biological functions through protein-protein interactions. Due to their small size, membrane microproteins may be uniquely suited to act as singular protein domains that engage with much larger proteins to fine-tune very complex biological systems (Figures 1 and 2). To date, biological functions have only been assigned to a very small fraction of predicted microproteins, which indicates that an enormous amount of interesting biology remains hidden amongst these uncharacterized proteins and the elucidation of their functions will likely drive new frontiers in membrane protein regulation and biology. Additionally, as many membrane proteins are current therapeutic targets, the concept of modulating the function of these membrane proteins from within the bilayer through the selective manipulation of membrane microproteins holds a lot of exciting potential therapeutic promise.