The Endoplasmic Reticulum

The endoplasmic reticulum (ER) accounts for more than 50% of the total cellular membrane. The ER is composed of a highly organized network of tubules and cisternae, extending through the cytoplasm. The ER membrane is continuous with the outer membrane of the nucleus. Moreover, the ER interacts with microtubules, and this

Figure 4: Translocon-associated proteins. The ribosome starts the translation of mRNA coding for secreted and membrane proteins in the cytosol (1). The signal sequence (red circles) is recognized by the Signal Recognition Particle (SRP, in green, 2), which drives the ribosome/mRNA/nascent polypeptide complex to the SRP-receptor (SRP-R), at the level of the ER, and more particularly close to the translocon machinery (red channel, 3). There, the translation is resumed (4) and the signal sequence is removed by the Signal Peptidase, in the lumen of the ER (5). During protein synthesis, the OligoSaccharylTransferase (OST) adds a pre-existing carbohydrate chain from a lipidic dolichol pyrophosphate to an asparagine residue of the nascent polypeptide (6).

The main functions of the ER in mammalian cells are described in section 2.a.

Proteins leave the ER at the level of Endoplasmic Reticulum Exit Sites described in the section 2.b, and reach the Endoplasmic Reticulum to Golgi Intermediate Compartment (ERGIC), described in section 2c.

a) Main functions of the endoplasmic reticulum

The ER is the site of many crucial reactions within the cells. As described in the first part of this chapter, the rough ER is the site of cotranslational insertion of soluble and transmembrane proteins destined to the exocytic pathway, and the place where they are glycosylated. The glycan moieties are also modified in the ER. It is also the place where nascent polypeptides are folded and assembled, with the help of many molecular chaperones. Enzymes including Peptidyl Propyl cis/trans Isomerases (PPIases) or members of the Hsp70 family (Heat-Shock Proteins 70) ensure the maintenance of a folding-competent state. Moreover, disulfide bond formation is catalysed by thiol:protein disulfide oxidoreductases and protein disulfide isomerases (PDIs) (Holtzman, 1997; Wilkinson & Gilbert, 2004).

The smooth ER is continuous with the rough ER but its membrane is devoid of ribosomes. It is the main site for synthesis of lipids notably phospholipids, glycolipids and cholesterol. In general, lipid biogenesis starts on the cytosolic face of the ER membrane, and the newly synthetized lipids are transferred into the luminal face of the ER membrane, with the help of flippases. Lipids are then transported along the secretion pathway.

Finally, the ER is the main calcium ion store of the cell, with a Ca²⁺ concentration 1’000 to 10’000 times higher than the cytosol. Calcium plays a key role in many intracellular signalling events and controls many cellular processes. A large part of intracellular signalling events thus involve the ER, and the control of calcium fluxes in and out of this organelle.

b) Endoplasmic Reticulum Exit Sites (ERES)

Once synthesized and correctly folded, proteins are allowed to progress through the secretion pathway. The exit out of the ER of correctly folded proteins takes place at specific sites called Endoplasmic Reticulum Exit Sites (ERES) or transitional ER (tER) (L.

Orci et al., 1991; Palade, 1975). At these sites, the ER membrane is coated with COPII components (the cytosolic coat of vesicles responsible for anterograde transport, as described in the second part of this manuscript). In mammalian cells, these sites appear by fluorescence microscopy as largely immobile and long-lived punctae associated with the ER (Hammond & Glick, 2000; D. J. Stephens, Lin-Marq, Pagano, Pepperkok, &

Paccaud, 2000).

The number and size of ERES can vary, depending on the cargo load that they have to deal with (Farhan, Weiss, Tani, Kaufman, & Hauri, 2008), underlining the plasticity of these structures. Secreted and membrane proteins are concentrated and then packaged into COPII-coated vesicles at the ERES and allowed to exit the ER. The next step of their journey to the plasma membrane is the passage in the Endoplasmic Reticulum to Golgi Intermediate Compartment (ERGIC).

c) The Endoplasmic Reticulum to Golgi Intermediate Compartment (ERGIC)

First described in 1984, the ERGIC, also known as the Vesiculo-Tubuluar Cluster (VTC) was initially proposed to be either a specialized sub-domain of the ER (Sitia &

Meldolesi, 1992) or of the Golgi complex (Mellman & Simons, 1992), or an independent organelle within the cell. The ERGIC notably exhibits a different biochemical composition from both the ER and the Golgi (Schweizer et al., 1990). Its main roles are to concentrate the anterograde cargo proteins, and to control their quality and their sorting.

The ERGIC is identified by a high concentration of the mannose-binding receptor ERGIC-53. This protein is described later in this manuscript (See section C.2. c. 1). Both COPI and COPII coat components (described below) are found at the level of the ERGIC, suggesting that the ERGIC forms a platform for COPII-driven anterograde transport, as an intermediate to the route to the Golgi complex, and COPI-driven retrograde transport.

But it is still under debate whether the ERGIC is a transient compartment rather than a stable compartment. Two models are today considered, the maturation model versus the stable compartment model (Figure 5).

- In the maturation model, the ERGIC is formed by the fusion of COPII-coated vesicles emanating from the ER. These transient clusters undergo homotypic fusion, and mature to eventually give rise to the cis-Golgi.

However, this model is mostly based on the observation of the transport of an overexpressed viral protein, the thermosensitive mutant of the G protein from vesicular stomatis virus (tsO45-VSV-G) (D.J. Stephens &

Pepperkok, 2001).

- In the stable compartment model, ERGIC receives lipids and proteins from the ER by COPII-coated vesicles forming an independent, stable organelle (C. Appenzeller-Herzog, 2006). Proteins are then thought to be transported to the Golgi complex possibly via COPI-mediated transport.

Figure 5: the ER-to-Golgi Intermediate Compartment. The ERGIC can be seen as a transient compartment (upper panel), in which COPII-coated vesicles (red coat) coming from the ER fuse between each other. This transient compartment then matures to eventually become the cis-Golgi itself. In the stable compartment model (lower panel), COPII-coated vesicles fuse and give birth to the ERGIC, as a stable compartment. From there, COPI-coated vesicles (green coat) transport lipids and proteins from the ERGIC to the cis face of the Golgi complex.

As mentioned before, the ERGIC is also the first sorting platform after ER exit.

Escaped, ER-targeted proteins are recycled back via COPI-coated vesicles (Gaynor, Graham, & Emr, 1998; Letourneur et al., 1994; Pelham, 1994). On the contrary, secreted and membrane proteins are allowed to continue their journey to the Golgi complex.

In yeast, no ERGIC compartment has been formally described.