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# Insertion of membrane proteins into lipid membranes

Integral membrane proteins are ubiquitous throughout living organisms, ranging from prokaryotes to mammals, accounting for approximately 20-30% of all proteins (Wallin et al. 1998). They perform a diverse set of functions ranging from signal transduction, to ion transport or even photosynthetic reaction centers. While their activity might vary dramatically, all these proteins experience a similar challenge. They must traverse the amphiphilic lipid membrane to reach their correctly folded state. The ways in which nature has overcome this challenge will be the primary focus of this page.

# Features and diversity of membrane proteins

Prior to discussing the mechanisms of membrane insertion, it is important to characterize key features of transmembrane proteins and their topology.

#### Characteristics of transmembrane domains

A transmembrane domain (TMD) is defined as a region of a polypeptide chain that completely traverses the hydrophobic region of the bilayer. The most common TMD’s are 20 amino acids long and form a tightly coiled structure known as an α-helix. An α-helix is the preferred structure as it maximizes hydrogen bonding within the backbone of the chain, effectively shielding the hydrophilic regions of the amino acid backbone from the surrounding acyl chains of the lipids. An integral membrane protein (IMP) is composed of one or more TMD’s. When studying an integral membrane protein’s sequence, it is possible to identify the TMDs by generating a hydrophobicity plot. The TMDs will correspond to regions of amino acids that are hydrophobic (Kyte & Doolittle 1982). However, the determination of TMDs through this method is not always accurate. The hydrophobicity plot of the G-protein coupled receptor, bovine rhodopsin, shown in Figure 1b shows a region that is known to be a transmembrane domain even though it has several hydrophilic residues within it which makes its identification through this analysis very challenging.

Figure 1: Structural diversity of membrane proteins (Shao & Hegde 2011).

#### Determinants of topology

The topology of an IMP refers to the orientation of the protein in the membrane. A protein can be in either the type I or type II topologies. These topologies are illustrated in Figure 1a. Type I indicates that the N-terminus of the protein passes through the membrane first. Type II topology implies that the N-terminus does not cross the membrane, causing the later segments of the protein to pass through first instead. The driving force to chose one conformation over another is composed of several additive features of the protein. The most prominent effect, at least for bacteria, comes from the positive inside rule. The positive inside rule states that positively charged amino acids, such as arginine and lysine, will not cross the membrane and remain in the cytosol (Hatmann et al. 1989). This is dictated by the strong positive charges interacting repulsively with the channel that I will describe later in this page. In bacteria the asymmetry between the leaflets and the strong proton motive force across the membrane cause the cytosolic side of the membrane to be far more favorable for positive amino acids compared to the periplasmic side (van Klompenburg 1997).

# Co-translational membrane protein insertion

Proteins can be inserted into membranes in several different ways, but the most prevalent method is through simultaneous insertion of the protein as it is translated. This is achieved in two distinct steps. First the translation machinery must identify that a given protein is a membrane protein. Next the protein must be passed through the membrane. The predominant way that this is done across all studied organisms is through a protein conducting channel, referred to as the Sec translocon.  These two major steps will be detailed below.

#### Recognition and targeting

Recognition occurs on the ribosome as the nascent polypeptide chain emerges from the exit channel by the Signal Recognition Particle (SRP). This protein has a nonspecific hydrophobic motif that interacts with hydrophobic amino acids as they are translated. Consecutive hydrophobic residues lead to a strong interaction with the SRP and causes the ribosome to stall translation. The SRP-riboosome complex then diffuses around the cytosol until it interacts with the membrane bound receptor specific to the bound SRP. This protein, either the eukaryotic SRP Receptor (SR) or bacterial FtsY, binds and then shuttles the SRP-ribosome complex to the Sec translocon. Once the SRP-ribosome-Sec complex is made, the nascent polypeptide chain is transferred to the Sec translocon for insertion into the membrane. This process is depicted in Figure 2. The critical aspect of this process is that it prohibits the hydrophobic region of the polypeptide from exposure to the hydrophilic cytosol to avoid misfolding and aggregation (Grudnik et al. 2009).

Figure 2: General steps of cotranslational membrane protein insertion (Shao & Hegde 2011).

#### Sec translocon

In both prokaryotes and eukaryotes, there are protein conductive channels generally referred to as Sec translocases. The prokaryotic version SecYEG and the eukaryotic version Sec61 are both heterotrimers with similar structure and function. These channels have two fundamental features that enable the insertion of proteins into the membrane. They contain a hydrophilic channel that allows hydrophilic residues of a polypeptide to pass through the membrane. They also contain a lateral gate that opens to expose the interior of the channel to the lipid acyl chains. This allows for hydrophobic residues to enter through the channel and then interact directly with the lipid tails while avoiding the polar head groups. These channels therefore allow the nascent chain that is being translated to effectively “thread” in and out of the membrane as many times as is required to reach the final structure. (van den Berg et al. 2004, Cymer et al. 2015). Upon complete insertion an alpha-helical domain referred to as the "plug" moves into the channel to prohibit other species from pass through. For relatively simple transmembrane proteins such as single spanning proteins, these channels are sufficient. However, when the protein contains many domains or does not adhere to the guidelines of standard membrane protein, other accessory factors are required to ensure correct insertion and folding.

Figure 3: The Sec61 translocon and potential accessory factors (Shao & Hegde 2011).

# Accessory factors

While the translocase complex is the primary requirement for most membrane proteins, more complex IMPs require accessory factors to aid in its insertion and maturation. Below is a breakdown of different accessory factors for prokaryotes and eukaryotes.

#### Eukaryotes

Numerous other membrane proteins have been found to interact with the Sec translocon complex in eukaryotes, however their necessity remains unclear. Enzymes like the signal peptidase and the oligosaccharyl transferase have been found to aid in the folding and maturation of certain IMPs by either cleaving or glycosylating respectively (Yamagisi et al. 2011). The translocon-associated protein (TRAP) complex may help fortify the ribosome-translocon complex. The translocating-chain associating membrane (TRAM) protein is currently expected to insert TMDs with weak hydrophobicity and therefore is only required for certain IMPs. (Heinrich et al. 2000)

#### Prokaryotes

As in eukaryotes, there are several accessory factors that have been found to aid in the insertion of IMPs in prokaryotes. SecA is an ATPase that has been shown to be essential to push highly hydrophilic proteins through the Sec channel (Zimmer et al. 2008). The YidC insertase has been shown to be aid in the lateral partitioning of IMPs out of the channel into the membrane. Its role is expected to be that of a foldase which helps with the formation of α-helices (Beck et al. 2001). It has also been found that the presence of the phospholipid phosphatidylethanolamine (PE) is required for the folding of certain IMPs. As PE is a positively charged lipid, it interacts with acidic residues that flank the TM and stop the residues from wanting to translocate to the positively charged periplasmic side of the membrane (Bogdanov et al. 2008).

## Post-transational Membrane Protein Insertion

Although the vast majority of proteins are inserted into the membrane cotranslationally, some proteins are inserted after full translation. Some IMPs, such as Tail-Anchored (TA) proteins, are unable to enter the standard path of integration. TA proteins are membrane proteins that have a long hydrophilic N-terminal sequence prior to their TMD. These proteins are difficult for the SRP unable to identify as IMPs as they rely on the binding of the TMD. This necessitates other methods of insertion. Eukaryotes and prokayotes have addressed this problem differently. The most prevalent methods are detailed below.

#### Eukaryotes

TRC40 and GET pathway

Insertion of TA proteins in mammalian cells utilizes a cytosolic ATPase named the TMD recognition complex of 40kDa (TRC40). The exact mechanism of this complex is still unclear, but the complex functions as a chaperone that binds to TMD post translation and then carries the protein to a docking complex embedded in the ER membrane which allows passage of the TMD into the membrane (Stefanovic & Hegde 2007, Casson et al. 2017). The pathway in yeast parallels the TRC40 complex but utilizes proteins named Get1/2/3 (Schuldiner et al. 2008)

TMD recognition

It has been observed that neither the TRC40 or GET proteins are capable of identifying a TMD on their own. They utilize a pretargeting complex that docks on ribosomes similar to the SRP. The pretargeting complex does not, however compete with the SRP to sit at the exit site of the ribosome. Instead it binds distal to the exit site. The hydrophobic pocket of the pretargeting complex is much larger than that of the SRP and is able to accommodate a hydrophobic alpha helix of about 20 amino acids in length. Once the pretargeting complex binds to a TMD, it diffuses away from the ribosome and searches for either Get3 or the TRC40 complex in yeast or mammalian cells respectively. Once they interact, the TA protein is transferred to TRC40/Get3 to then be chaperoned to the ER membrane (Mariappan et al. 2010, Wang et al. 2010).

Figure 4: Schematic of the post-translational TA protein insertion pathway in eukayotes (Shao & Hegde 2011).

#### Prokaryotes

YidC Insertase

Certain small prokaryotic IMPs were long believed to insert spontaneously as it was found that they did not rely on the presence of the Sec translocon. It was later found however that there was another translocase, named the YidC insertase, that was responsible to catalyzing the "spontaneous" insertion. The requirement of this insertase is highly evident for the insertion of the Pf3 coat protein as well as the subunit c of the F1F0 complex and one of the subunits of the cytochrome bo oxidase. The YidC insertase primarily works through the insertion of two hydrophobic alpha helices forming a helical hairpin into the membrane. This insertion process is depicted in Figure 5. YidC has also been found to assist the SecYEG translocon in the stabilization and helical packing of multispanning IMPs. As the alpha helices laterally diffuse out of the Sec translocon they interact with the YidC insertase and form helicial hairpins within the membrane. This helps promote the exit of the TMD out of the channel and interact with adjacent TMDs (Xie & Dalbey 2013).

Figure 5: Insertion of a helical pin into the membrane by the YidC insertase (Xie & Dalbey 2013).

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