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 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.
Eucaryotic cells use the principle of compartmentalization to streamline the flow of information within the crowded intracellular environment. Different subcellular compartments have occurred during evolution as the result of either endosymbiosis, invagination of the plasma membrane, budding off from other previously formed organelles, or, as discussed more recently, from the autogenous fusion of plasma membrane protrusions 
. Irrespective of its inside-out (protrusions of the procaryotic plasma membrane) or outside-in (invagination of the procaryotic plasma membrane) origin, the lumen of the endoplasmic reticulum (ER) was at first similar to the extracellular milieu and therefore different from the cytosol. In mammalian cells, the ER lumen still reflects its extracellular derivation based on the high concentration and storage of calcium 
. Next to the nucleus, the ER is one of the largest organelles in many cell types and is abundantly present in secretory cells such as those found in the endo- and exocrine portions of the pancreas 
. The correlation between protein secretion and abundance of the ER is not coincidental and substantiates the importance of the ER for this process 
. Indeed, the ER represents the entry point for proteins to the secretory pathway including the exocrine zymogens and endocrine hormones released by the pancreas.
About one-third of eucaryotic genes encode for polypeptides that require targeting to the ER membrane 
. Those polypeptides belong to soluble, membrane-associated, or integral membrane proteins that are all handled and distributed by the ER. Although this organelle represents a vast network that spans from the outer nuclear membrane to the periphery, newly synthesized proteins do not find the ER autonomously. Instead, precursor polypeptides rely on specialized targeting mechanisms that direct them to the ER membrane (Figure 1
) in a co- or post-translational fashion 
. In the case of co-translational protein targeting, a nascent polypeptide is recognized during the process of ribosomal translation. As soon as a specific amino acid stretch of the precursor, a so-called targeting signal, emerges from the ribosomal exit tunnel, it is recognized by a targeting factor that directs the complex of ribosome and nascent chain to the ER membrane. The signal recognition particle (SRP) was the first co-translationally acting targeting factor that was discovered and shown to target the ribosome-nascent chain complex (RNC) with the help of the cognate SRP receptor (SR) to the ER membrane 
. In contrast, post-translational protein targeting occurs after a precursor is fully synthesized and released from the ribosome into the cytosol. Key features of post-translationally targeted polypeptides can be a short overall precursor length (<100 amino acids), a C-terminal positioning of a transmembrane helix (TMH) that serves as a targeting signal, or, as is the case for yeast, a “weak” N-terminal signal peptide (SP) that is required for targeting 
. These features prevent efficient recognition by the co-translational targeting factor SRP and require the presence of a post-translational targeting factor such as GET3. GET (guided entry of tail-anchored proteins) is the acronym for guided entry of tail-anchored proteins. This implies that GET3 is involved in the targeting of tail-anchored (TA) proteins, substrates that carry a single C-terminally located TMH as a targeting signal. In addition, GET3 has been shown to promote the targeting of short precursor polypeptides with a cleavable N-terminal SP 
. Recent evidence has uncovered another SRP-independent (SND) targeting pathway that seems to function as a backup system with some substrate spectra that overlap with the GET and SRP pathway 
. Additionally, abundant cytosolic chaperones support the targeting of fully-synthesized precursor polypeptides, but they are not discussed here in detail 
Figure 1. 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 . Abundance values for the receptor components are based on the quantitative mass spectrometry of mammalian cells . 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 . 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.
After targeting to the ER membrane, co- and post-translationally arriving polypeptides rely on one of the protein translocation machineries residing in this membrane. Similar to the diversity of precursor polypeptides that are handled by multiple targeting pathways, multiple protein translocases have also evolved to support the insertion or translocation of proteins into or across the ER membrane 
. As the ER membrane originated from the procaryotic plasma membrane, some of the translocation machines of the ER resemble their bacterial ancestors 
. The first ER protein translocase that was described and studied in much detail was the Sec61 complex 
. This translocase can open up a “fenestrated” conduit through the ER membrane for the translocation of SP-carrying proteins as well as the lateral release of TMHs 
. Other precursor polypeptides, such as TA proteins, can make use of alternative translocation machines such as the GET1/2 complex or the ER membrane protein complex (EMC) 
. Interestingly, precursor proteins relying on the GET1/2 complex or EMC require a differentially shaped opening compared to the membrane-spanning pore that is provided by the Sec61 complex. This shows that nature has apparently found more than one solution for polypeptides to traverse a membrane.
It is now half a century since Blobel and Sabatini published their first “tentative scheme” on what they would later coin the signal hypothesis. They speculated about the “role of the ribosome-membrane interaction in the vectorial discharge of proteins into the ER”, which “is indicated by (a) the close association of the nascent polypeptide chain with the ER membrane and (b) the close association of the large ribosomal subunit with this membrane” 
. As no one size fits all, several types of targeting signals, targeting pathways, and protein translocases have since then been described, which demonstrate the diversity of transferring proteins from the cytosol to the secretory pathway. However, in light of the macromolecular crowding and potential off-target destinations, one underlying theme that unifies those functional targeting and translocation networks is “safety first”. During their journey from the inside of the ribosome to the ER membrane, the nascent polypeptides are handed over from one protected compartment and cavity to the next to avoid extensive folding and premature, unproductive interactions. After leaving the shielded environment provided by the ribosome, hydrophobic targeting signals are cradled by a binding pocket of one of the targeting factors before they are transferred to a narrow opening of one of the translocation machineries that acts as a membrane-integrated chaperone.
2. Different ER Protein Translocases Act as Membrane-Integrated Chaperones
Consistent with the multiplicity and complexity of targeting signals and targeting pathways described in the previous sections, recent findings in the field of ER protein import have also extended this concept to a small assortment of ER protein translocases. Different types and arrangements of protein translocases, sometimes acting in concert, manage the translocation or insertion of a dedicated subset of incoming polypeptides (Figure 2
). The different multimeric protein complexes that catalyze the insertion of an unfolded polypeptide, nascent or full-length, behave in a way very similar to targeting factors acting as a temporary safe harbor. As such, a membrane-integrated protein translocase transiently shields segments of incoming cargo and thereby facilitates the partitioning of hydrophobic TMH into the ER membrane or the translocation of soluble domains into the ER lumen. Thus, incoming polypeptides are handed over from a soluble targeting factor to a membrane-integrated translocase, both of which provide a chaperone-like environment and prevent improper interactions and the aggregation of unfolded polypeptides. Herein will summarize the central constituents of protein translocase complexes and refer the reader to the special issue on the “Mechanisms of ER Protein Import” as well as others