The endomembrane system constitutes a group of membranes and organelles found in eucaryotic cells. It includes the cell membrane, the nuclear membrane, vacuoles, lysosomes, the Golgi apparatus, vesicles and the endoplasmic reticulum (ER). The membranous organelles interact together to synthesize and transport proteins, transport and metabolize lipids and to detoxify posions. The organelles communicate with one another and are connected directly or through transport vesicles.
The Endoplasmic reticulum is a membranous system of interconnected tubules and flattened saccules called cisternae that is located in direct physical continuity with the nuclear envelope. In animal cells, the Endoplasmic Reticulum accounts for more than half the total membrane content. The Endoplasmic reticulum encloses a cisternal space known as the ER lumen. The cisternal space is continuous with the perinuclear space but separate from the cytosol. The perinuclear space is the compartment between the inner and outer nuclear membranes. Two distinct but interconnected regions of the endoplasmic reticulum exist, namely the Rough Endoplasmic Reticulum (RER) which is characterized by the presence of membrane-bound ribosomes attached to its cytoplasmic surface and the Smooth Endoplasmic Reticulum which lacks ribosomes.
Transport into the Endoplasmic Reticulum is co-translational in that the protein inserted into the endoplasmic reticulum as it is being synthesized by the ribosome and in that the two processes are linked together. This contrasts with protein transport into the nuclei, mitochondria, chloroplasts, and peroxisomes which occurs post-translationally. Transport into the Endoplasmic reticulum also requires that the protein be unfolded. The protein folds once it enters the lumen. Protein transport into the Endoplasmic Reticulum is unidirectional in that the protein does not return into the cytosol unless improper folding of the protein occurs.
Proteins destined for the endomembrane system express a signal sequence known as the ER signal sequence. This sequence is located at the N-terminal end of the protein and consists of at least 8 hydrophobic amino acids. Proteins that contain no signal peptide at their N-terminal end are translated on ribosomes that are free in the cytosol. These proteins either remain in the cytosol or are transported to the nucleus, peroxisomes, chloroplasts, or mitochondria. Proteins containing a signal sequence result in the binding of their ribosome to the membrane of the endoplasmic reticulum and the insertion of said protein into the rough endoplasmic reticulum. Soluble proteins become fully translocated into the cisternal lumen. Transmembrane proteins, on the other hand, are only partially translocated and are inserted into the ER membrane to reside or are destined to the plasma membrane or other organelles. Proteins that are retained in the ER lumen possess a common sequence of four amino acids at their COOH terminal domain, namely the KDEL sequence.
As previously mentioned, targeting to the endoplasmic reticulum occurs by virtue of the interaction between the ER signal sequence and its receptor, namely the signal recognition particle (SRP). Once the signal recognition particle binds to the ER signal sequence and the ribosome, a pause in protein translation occurs and the ribosome docks to the SRP receptor. The interaction of the signal peptide with the SRP receptor leads to the stimulation of GTPase activity present in both the signal sequence and the SRP receptor. Upon docking, the signal peptide and the SRP receptor hydrolyse their bound GTPs, as a consequence of which the SRP receptor is ultimately released, the translation pause is eased, and translocation of the protein occurs whereby the growing N-terminal end of the protein passes into the lumen of the ER. Translocation occurs by virtue of a heterotrimeric protein-containing channel, namely the Sec61 translocon. The signal peptide enters the ER lumen and is proteolytically cleaved by a membrane-bound signal peptidase. Post-translational modifications of the protein occur by virtue of enzymes in the ER lumen. These modifications include the addition of N-linked carbohydrates to glycoproteins and disulfide bond formation, processes which promote protein folding in the ER lumen.
As previously mentioned, soluble proteins pass completely into the membrane. Once the soluble peptide has reached 150 residues, a signal peptidase cleaves off the signal sequence, which for soluble proteins, exists at the N-terminus. The newly synthesized protein is released freely in the ER lumen. The insertion of transmembrane proteins into the Endoplasmic Reticulum lumen differs depending on the orientation of their transmembrane domains across the lipid bilayer. For single transmembrane proteins, two possibilities can arise. Firstly, a transmembrane protein with its N-terminal end on the lumen side of membrane but its carboxylic end in cytoplasm can arise if the signal peptide is N-terminal end, and the signal peptide is then excised but another 8-14 hydrophobic amino acid residue sequence (known as a stop-transfer sequence) in the middle of the peptide which is not cleaved off stops translocation. Translation, then, continues in the cytoplasm. The N-terminal end is cleaved off and released into the lumen and the protein conserves its orientation with its C-terminal end outside and N-terminal end inside. Alternatively, a transmembrane protein with its carboxyl end on the lumen side of membrane but its N-terminal end in cytoplasm can result. This can happen if the signal peptide sequence is not located at the N-terminal end but rather is located internally in the middle of the protein. The signal peptide is, as a result, not cleaved. It becomes a transmembrane domain of the protein. For multipass transmembrane proteins, a start transfer sequence initiates translocation which proceeds until a stop transfer sequence is reached. An alternating series of start and stop sequences stitch the membrane proteins into the lipid bilayer.
One of the co-translational modifications that proteins undergo in the endoplasmic reticulum includes N-linked glycosylation. In N-linked glycosylation, pre-formed oligosaccharides (which include 2 N-acetylglucosamines, 9 mannoses and 3 glucoses) are assembled on a lipid carrier (dolichol) and attached by oligosaccharyltransferase to asparagine residues of the nascent polypeptide chain. The enzymatic removal of three glucose residues and one mannose residue is then accomplished. The dolichol-linked oligosaccharide is, essentially, assembled on the cytosolic face of the ER, flipped to the lumen, and transferred to the Asn-X-Ser/Thr consensus sequence.
Calnexin, a chaperone protein which is bound to the endoplasmic reticulum, binds to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides (monoglucosylated oligosaccharide). Removal of the terminal glucose by a glucosidase releases the protein from calnexin. If the protein is still improperly folded, a glucosyl transferase enzyme transfers a new glucose from UDP-glucose to the N-linked oligosaccharide, retaining the protein in the Endoplasmic reticulum.
GPI anchoring of proteins is a post-translational modification for anchoring of the protein to the outer surface of the plasma membrane. Proteins destined to be GPI-anchored are translocated to the lumen of the ER. The GPI attachment signal peptide at the C-terminus is cleaved off and the preassembled GPI is transferred to the last amino acid of the C-terminal end. The core backbone of the GPI anchor consists of PI, glucosamine, three mannose and ethanolaminephosphate.
Soluble, membrane proteins and lipids that are produced in the ER are subject to a vectorial (cis to trans) flow through the Golgi apparatus. The major types of coat proteins used in vesicle formation are COPII, COPI, and clathrin. COPII coat proteins form the vesicles that move from ER to Golgi. COPI coat proteins are used between parts of the Golgi apparatus as well as to form vesicles going from the Golgi apparatus back to the ER. Clathrin is used to form vesicles leaving the Golgi for the plasma membrane as well as for vesicles formed from the plasma membrane for endocytosis.
The KDEL receptor ensures that soluble proteins are retained in the endoplasmic reticulum. The KDEL receptor targets any protein displaying the KDEL sequence into COPI coated retrogate vesicular transport. binds its ligands in the Golgi apparatus, where it captures proteins that have escaped the ER, so that it can return them. The KDEL receptor binds its ligand more weakly in the ER, but more strongly in the Golgi apparatus so that those soluble proteins present at low concentration in the Golgi apparatus can be returned to the rough Endoplasmic reticulum that houses a high concentration of KDEL containing proteins.
Protein processing within the Golgi apparatus involves the synthesis and modification of carbohydrate portions of glycoproteins. A major process involves the modification of N-linked oligosaccharides that were added to proteins in the ER . As previously mentioned, proteins are modified within the ER via the addition of an oligosaccharide consisting of 14 sugar residues. Three glucose residues and one mannose are then removed in the ER. Following transport to the Golgi apparatus, N-linked oligosaccharides are processed within the Golgi apparatus. The first modification involves the removal of three additional mannose residues. This is followed by the sequential addition of an N-acetylglucosamine, the removal of two more mannoses, and the addition of a fucose and two more N-acetylglucosamines. Finally, three galactose and three sialic acid residues are added.
The processing of the N-linked oligosaccharide of lysosomal proteins differs from that of secreted and plasma membrane proteins. In place of the initial removal of 3 mannose residues, proteins targetted for lysosomes undergo mannose phosphorylation. In this process, N-acetylglucosamine phosphates are added to specific mannose residues. This is followed by removal of N-acetylglucosamine, which leaves mannose-6-phosphate residues on the N-linked oligosaccharide. The phosphorylated residues are not removed during further processing. Instead, they are specifically recognized by a mannose-6-phosphate receptor in the trans Golgi network, which directs their transport to lysosomes.