The Molecular Biodiversity of Protein Targeting and Protein Transport Related to the Endoplasmic Reticulum

Abstract

Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades taught us one humble lesson: no one size fits all. Cells use an impressive array of components to enable the safe transport of protein cargo from the cytosolic ribosomes to the endoplasmic reticulum. Safety during the transit is warranted by the interplay of cytosolic chaperones, membrane receptors, and protein translocases that together form functional networks and serve as protein targeting and translocation routes. While two targeting routes to the endoplasmic reticulum, SRP (signal recognition particle) and GET (guided entry of tail-anchored proteins), prefer targeting determinants at the N- and C-terminus of the cargo polypeptide, respectively, the recently discovered SND (SRP-independent) route seems to preferentially cater for cargos with non-generic targeting signals that are less hydrophobic or more distant from the termini. With an emphasis on targeting routes and protein translocases, we will discuss those functional networks that drive efficient protein topogenesis and shed light on their redundant and dynamic nature in health and disease.

Keywords: EMC; GET; SND; SRP; Sec61 complex; endoplasmic reticulum; hydrophobicity; positive-inside rule; protein targeting; protein transport; signal peptide; transmembrane helix.

Figure 1

Features of human signal peptides and the amino acid distribution in key positions. (A) The tripartite segmentation of a cleavable signal peptide (SP) found at the N-terminus (NH₃⁺) of secretory proteins. Characteristic attributes of the three sub-domains are given below. (B) A histogram showing the SP length distribution of 3584 human SP annotated at Uniprot. Numbers at the top of the diagram represent frequencies of the highlighted area. Almost three-quarters of SPs are 21–40 amino acids (aa) in length. (C,D) Histograms showing the frequencies of the twenty proteinogenic amino acids in the second (C) of twelve (D) positions of the same human SPs considered in (B) ordered from the lowest to the highest fraction. Amino acids are listed on the x-axis according to the one-letter code. The acidic amino acids aspartate (D) and glutamate (E) are labeled in red and the basic amino acids arginine (R) and lysine (K) in blue.

Figure 2

Length, charge, and topology of transmembrane helices of human bi- and polytopic membrane proteins. (A) The histogram shows the relative fraction of bitopic membrane proteins (MPs) with one transmembrane helix (TMH) and polytopic MPs with two or more TMHs. The data are based on the 20,056 TMHs of 4853 human MPs annotated at Uniprot. (B) Grey bars show the average length (left y-axis) with standard deviation of all first, second, and later TMHs till the twelfth TMH. White numbers at the bottom of the bar indicate the number of TMHs found in the human MPs. The blue diamond represents the relative fraction of the same TMHs that do not carry any charged residues (right y-axis). (C) Classification of bi- and polytopic (_pt) membrane proteins based on topology, presence of a signal peptide (SP), and localization of a TMH within the primary structure. SPs are colored according to Figure 1 and their cleavage by the signal peptidase is indicated by scissors. For orientation purposes, the C-terminus (C) of each representative is indicated. Black lines indicate domains up- and downstream of a TMH. Cis- and trans-acting factors that influence the final topology of MPs are mentioned on the right and are further described in the text. aa, amino acids; ave., average; w/o, without.

Figure 3

Major components and hallmarks of the mammalian SRP, SND, and GET targeting pathways. The top half shows a graphical output of the major components that shape the three targeting pathways SRP, SND, and GET. The dotted lines indicate a zoomed-in view of the BAG6 pre-targeting complex cooperating with SGTA and other cellular components. The double-headed arrow suggests the cycling of TA proteins between the two chaperones SGTA and BAG6. The bottom half summarizes some of the key features that differentiate the pathways from each other. Ribosome-associated and cytosolic targeting components are shown in grey colors and the cognate membrane receptors in shades of blue. Components (hSnd1, hSnd3) shown with a hatched color fill have not yet been identified in higher eucaryotes. Their existence is based on findings from yeast and the conserved nature of targeting machineries [21]. Abundance values for the receptor components are based on the quantitative mass spectrometry of mammalian cells [132]. Please note, there is considerable controversy about the abundance of GET1 and GET2 with some sources finding GET2 in four- to sevenfold molar excess over GET1 [133,134]. The N-terminus (N) of newly synthesized polypeptides is shown to accentuate the positioning of signal peptides (SPs) and transmembrane helices (TMHs) of different types of cargos, including tail-anchored (TA) proteins and glycosylphosphatidylinositol (GPI)-anchored proteins. BAG6, BCL2-associated athanogene 6; GET, guided entry of TA proteins; HSPs, heat shock proteins; SGTA, small glutamine rich tetratricopeptide repeat co-chaperone alpha; SND, SRP-independent; SR, SRP receptor; SRP, signal recognition particle.

Figure 4

The impact of ribosome-binding proteins and the ribosomal exit tunnel geometry on the nascent chain and targeting decision. The nascent chain (red line) is born at the peptidyl transferase center (PTC) and folds in the vestibule of the lower ribosomal tunnel after passing the constriction sites build by the universally conserved ribosomal proteins uL22 and uL4 (dotted lines). Early recruitment of targeting factors involves the mRNA (blue line) and the presence and folding of the nascent chain while still residing inside the tunnel (blue and red arrow, respectively). Ribosomal surface proteins such as uL23 and uL29 (orange) which build common docking sites are mapped next to the exit port. Moreover, eL39 (dotted ellipse) that lines the interior of the ribosomal tunnel and spans to its surface might contact the nascent chain and putative targeting factors. Similarly, NAC reaches deep into the tunnel where it scans nascent chains to coordinate between the various factors competing for the emerging nascent chain (black arrows). Accordingly, NAC may regulate the targeting priority of targeting factors including SRP, the BAG6 complex, SGTA, or the putative hSnd1 (hatched color fill) to ensure the targeting of different types of cargo. Note that NAC and SRP were mapped at overlapping sites next to the tunnel, while the location of alternative factors remains unknown. BAG6, BCL2-associated athanogene 6; GET, guided entry of TA proteins; NAC, nascent polypeptide-associated complex; SGTA, small glutamine-rich tetratricopeptide repeat co-chaperone alpha; SND, SRP-independent; SRP, signal recognition particle.

Figure 5

Features of protein translocation machines and their substrate spectrum. Depicted from the left to right are targeting pathways (blue), membrane-integrated protein translocation machines (pink, purple), their favored types of clients (boxes), and some key features for the central components of the protein translocation machines. Apart from GET1/2, the membrane-anchored receptor components for the targeting pathways are not shown. The scarcely characterized SND pathway (grey) as an alternative targeting route for a subset of GPI-anchored, tail-anchored, short secretory, and polytopic membrane proteins is indicated once. If SND delivers substrates either to a preferred ER translocase or a different one, or if its membrane-embedded component(s) might perform a dual function as receptor plus insertase similar to the GET1/2 complex remains to be seen. The auxiliary TMCO1 and PAT complexes are presented as one assembly. Of note, the PAT complex that was also described as a stand-alone intramembrane chaperone complex, is a heterodimer comprised of the indicated proteins CCDC47 and Asterix [251]. The TMCO1 containing translocon comprises the Sec61 channel and five accessory factors: TMCO1, CCDC47, and the Nicalin–TMEM147–NOMO complex [252]. For reasons of simplicity, we refer to those five accessory factors as “TMCO1 complex”. The PAT and TMCO1 complex share at least one subunit, CCDC47. Further details of the TMCO1–PAT–Sec61 assembly and how they might act in concert to form an operational protein translocase can be found in the text. Signal peptides and transmembrane helices are integrated into the ER membrane and classified and colored according to Figure 1 and Figure 2. The cleavage of signal peptides for secretory and type I membrane proteins is indicated (scissors and cleaved signal peptides are shown) as is the C-terminus (C) for each type of membrane protein. The spectra of substrates preferentially handled by assemblies that entail the Sec61 complexes are represented by grey boxes. Substrates handled by the ER membrane complex (EMC) and the GET1/2 complex are highlighted by light blue and yellow boxes, respectively. Overlapping boxes depict overlaps in the substrate range. CCDC47, coiled-coil domain containing protein 47; NOMO, nodal modulator protein; TMCO1, transmembrane and coiled-coil domain-containing protein 1; TMEM147, transmembrane protein 147.

Figure 6

Disease associations of selected targeting and translocation components. The cartoon summarizes disease associations for critical subunits of targeting factors and translocation machines. Further details can be found in the text. ADPLD, autosomal dominant polycystic liver disease; ADSCN, autosomal dominant severe congenital neutropenia; ADTKD, autosomal dominant tubulo-interstitial kidney disease; CFT dysplasia, cerebro-facio-thoracic dysplasia; CVID, common variable immunodeficiency, ESCC, esophageal squamous cell carcinoma; IMNM, immune-mediated necrotizing myopathy; PCOS, polycystic ovary syndrome; RCC, renal cell carcinoma; THND syndrome, tricho-hepato-neuro-developmental syndrome.