Intracellular Compartments and Protein Sorting

1. Overview of the compartments of a eucaryotic cell.

• All eucaryotic cells have the same basic set of membrane-bound organelles (Fig 12-1) that are distributed not randomly in the cell.

• Nucleus à contains the genome and is the main site of DNA and RNA synthesis

• Cytoplasm à cytosol + organelles. The cytosol is the site of protein synthesis and intermediary metabolism.

• Endoplasmic reticulum (ER) à about ½ total membrane in a cell. Site of synthesis of integral membrane and secretory proteins, and of lipids for all membranes. Proteins are translocated into the ER from the cytosol during their synthesis, and hence the ribosomes on which they are made are tethered to the ER membrane (Rough ER). Proteins for other organelles are translocated there after their synthesis is complete. The ER also produces the lipid for the cell (Smooth ER), and is the main intracellular Ca²⁺ store.

• Golgi apparatus à receives lipids and proteins from the ER and sends them to various destinations, usually covalently modifying them en route.

• Mitochondria and (in plants) chloroplasts à ATP factories, respiration and photosynthesis. Contain their own genomes and ribosomes.

• Lysosomes à site of degradation of defunct organelles as well as macromolecules and particles taken in from outside the cell by endocytosis. Endocytosed materials first pass through endosomes.
• Peroxisomes à a.k.a microbodies, site of some specific oxidative reactions.

• The topological relationships of membrane-bound organelles can be interpreted in terms of their evolutionary origins (Figs 12-3, -4, -5).
  • Evolutionary evidence suggest the following grouping for the intracellular compartment in eucaryotic cells: (1) The nucleus and cytosol, which communicate through the nuclear pores and are thus topologically continuos. (2) All organelles that function in the secretory and endocytic pathway, i.e. ER, Golgi, endosomes, lysosomes, and other vesicles, in which their lumen is topologically equivalent to the extracellular medium. Note that the intermembrane space between the nuclear outer and inner membranes (perinuclear space) is continuos with the ER lumen. (3) The mitochondria, (4) the plastids (in plants only) and (5) the peroxisomes. For 4 and 5, the lumen and inner membranes are presumed to evolve from the cytosol and plasma membrane of an ancient bacterium.

• Proteins move between compartments in 3 different ways, all guided by sorting signals in the transported protein that a recognized by complementary receptor proteins in the target organelle (Fig 12-7).
  • Gated transport, between the cytosol and the nucleus through the nuclear pores
  • Transmembrane transport, membrane bound protein translocators directly transport specific proteins across a membrane from the cytosol into a space that is topologically different (e.g. ER membrane or lumen). The transported protein usually must unfold to snake through the membrane.
  • Vesicular transport, (next class) transport vesicles ferry proteins from one compartment to another.

• Two kinds of sorting signals, signal peptides and signal patches (Fig 12-8).
  • The signal peptide resides in a continuos stretch of amino acids (15-60 residues-long). It is often (but not always) removed from the mature protein by a signal peptidase once the sorting process is complete. Signal peptides direct proteins from the cytosol into the ER, mitochondria, chloroplasts, peroxisomes and nucleus; they are also used to retain luminal ER proteins. Signal peptides are easily transferred to a cytosolic protein thus, targeting it to the above compartments. (Table 12-3).
  • Signal patches are three-dimensional arrangements of atoms on the proteinís surface that form when the protein folds up. Since the atoms may come from different regions in the linear amino acid sequence, it is difficult to transfer signal patches onto cytosolic proteins. Signal patches identify regions on some proteins to be added specific sugar residues, which target them to the lysosomes.
  • It follows from the above that cytosolic proteins lack sorting sequences

• Panel 12-1, main experimental approaches to study protein sorting and targeting.

2. The ER

• The entrance of proteins into the ER represents a major branch point for the traffic of proteins within eucaryotic cells, corresponding to synthesis of proteins either on free ribosomes or membrane-bound ribosomes. Proteins destined for the cytosol or to be incorporated into the nucleus, mitochondria, chloroplasts, or peroxisomes are made on free ribosomes. In contrast, most proteins destined for secretion or incorporation into the ER, Golgi, lysosomes, or plasma membrane are synthesized on membrane-bound ribosomes and transferred into the RER as their translation proceeds.
• Free and membrane-bound ribosomes are functionally identical. Ribosomes engaged in the synthesis of secreted proteins are targeted to the ER by a signal sequence at the NH₂-terminus of the growing polypeptide chain. These contain a stretch of hydrophobic aas preceded by basic residues, about 20 residues in length à signal hypothesis, subject of a student presentation.
• Mechanism: As the SS emerge from the ribosome, they are recognized and bound by a signal recognition particle (SRP), consisting of six polypeptides and a small cytoplasmic RNA (7SL RNA). This binding inhibits translation and target the complex to the RER by binding to the SRP receptor on the ER membrane. SRP is then released and the ribosome binds to a protein translocation complex in the ER membrane; the SS is inserted into a membrane channel, translation is resumed and the unfolded growing polypeptide chain is translocated across the membrane into the ER. As translocation proceeds, the SS is cleaved by signal peptidase and the polypeptide is released into the ER lumen.
• Experiments of the kind outlined in Panel 12-1, as well as direct ion conductance measurements, have demonstrated that translocation occurs through aqueous channels formed by transmembrane proteins. Channels are opened by the SS and are maintained in the openconfiguration by ribosome binding. Both biochemical and genetic evidence point to the Sec61 complex of three transmembrane proteins, as a major component of the channel in yeast and mammalian cells. These proteins show high homology to the translocation machinery in E.coli.
• Insertion of Proteins into the ER membrane: proteins destined for incorporation into the plasma membrane or the membranes of the ER, Golgi, or lysosomes are first inserted into the ER membrane instead of being released into the ER lumen as above. Different integral membrane proteins differ in how they are inserted. Besides, one-pass and multiple-pass transmembrane proteins, some proteins have their N-terminus exposed to the cytosolic side, while yet others have their C-terminus so exposed. These orientations are established as the growing chains are translocated into the ER
• For one-pass proteins with their C-terminus exposed to the cytosol insertion into the membrane involves the sequential action of two distinct elements: a cleavable amino terminal SS that initiates translocation and a stop-transfer sequence that anchors the protein in the membrane.
• Proteins can also be anchored by internal signal sequences that are not cleaved by the signal peptidase. These are nevertheless recognized by SRP and targeted to the ER membrane as above. These SS act as stop-transfer sequences too. They can account for either orientation of the protein.
• Multi-pass transmembrane protein insert as a result of an alternating series of internal signal sequences and stop-transfer sequences, yielding loops in both sides of the ER membrane.
• Protein folding in the ER lumen: assisted by molecular chaperones like the BiP protein, a member of the Hsp70 family. BiP is thought to bind unfolded polypeptide chains, which upon only correct folding are released by BiP for further transport to the Golgi. In the ER, disulfide bond formation is promoted by an oxidizing environment and is facilitated by the enzyme protein disulfide isomerase.
• Protein glycosylation in the ER: N-linked glycosylation of proteins occurs in the ER while their translocation is still in progress. Oligosaccharide units of 14 sugar residues are added to acceptor Asn residues. Oligosaccharide is synthesized on a lipid (dolichol) carrier anchored on the ER membrane. It is then transferred to an Asn in the consensus sequence Asn-X-Ser/Thr by oligosaccharide transferase. 3 glucose and one mannose residues are removed.
• Glycophophatidylinositol (GPI) anchors: assembled in the ER membrane. Then added immediately after completion of protein synthesis to the C-terminus of some proteins by an exchange between the transmembrane region of the protein for the GPI anchor. These proteins are associated to the luminal (extracellular) side of the membrane only via their glycolipid.
• Smooth ER and lipid synthesis.
  • Glycerol phospholipids are synthesized in the ER membrane from cytosolic precursors. Two fatty acids linked to CoA carriers are first joined to glycerol 3-phosphate, producing phosphatidic acid, which is at the same time inserted into the membrane. A phosphatase converts PA into diacylglycerol (DAG) and to it, different head are added to form PC, PE, and PS. Phosphatidylinositol (PI) is formed from PA and not from DAG. Cholesterol and ceramide are largely made in the ER.
  • Because new phospholipids are only added to the cytosolic leaflet of the ER membrane, some of these must be transferred to the luminal leaflet by flippases to achieve a balance membrane growth. Transport of phospholipids between membranes of different compartments is mediated by water-soluble phospholipid exchange proteins (or phospholipid transfer proteins)(Fig 12-54).

3. Transport into and out of the nucleus

• Nuclear pore complex (NPC), contains ~ 100 different, mostly unknown proteins, some of which make up, ~ 9 nm-wide, aqueous channels, through which diffuse passively small molecules (<5 kDa) and proteins up to ~20 kDa. Larger molecules are actively transported in and out the nucleus through the pores, whose central channel can expand up to 40 nm. The central channel often shows a central plug of unclear function. On transcriptionally active or dividing cells, the traffic through the pores is high.
• Nuclear localization signal (NLS), located almost anywhere in the amino acid sequence, consisting of a short sequence (4-8 residues), rich in K and R, and usually contain P. The signals are thought to form loops on the protein surface. Proteins may contain more that one NLS.
• Protein import through the NPC can be operationally be divided into two steps, distinguished by whether they require energy. In the absence of ATP, proteins that contain NLSs bind to the NPC but do not pass through the complex. In this initial step, NLSs are recognized by a cytosolic receptor, and the receptor-substrate complex binds the pore. Two subunits make up this receptor, called importin: one subunit (importin-a) binds NLS, while importin-b, appears to bind the NPC. The second step, the actual translocation through the pore, requires ATP and GTP. The importin-a-NLS-containing protein complex is transported through the pore; importin-b dissociates during translocation. The small GTP-binding protein Ran is thought to promote the dissociation of the importin subunits.
• Recent studies have identified specific amino acid sequences, nuclear export signals (NESs), that account for the rapid export of some shuttling proteins from the nucleus to the cytoplasm. Like NLSs in nuclear import, NESs are thought to be recognized by receptors within the nucleus that direct outward transport trough the pores.
• The activity of some gene regulatory proteins is controlled by keeping them out of the nucleus until they are needed. The NLS of some of these proteins can be inactivated by phosphorylation. Others are bound to cytosolic proteins that either anchor them to the cytoskeleton in the cytosol or mask their NLSs. Upon stimulation, the inhibitory protein is released and the transcription factor is transported into the nucleus (Fig 12-19).

4. Transport into mitochondria and chloroplasts

• Most mitochondrial and chloroplast proteins are encoded in the nuclear genome and made in the cytosol. Mitochondrial proteins will be finally targeted to either the outer or inner membranes, or the intermembrane space, or the matrix. Besides the outer and inner membranes, and the intermembrane space, final destinations for chloroplast proteins include: the stroma and the thylakoid membrane and space (lumen)
• Most proteins are targeted to mitochondria by NH₂-terminal sequences of 15-35 residues, called mitochondrial targeting signals, that are removed by proteolysis following their import in the matrix. These sequences contain multiple positively charged and hydrophobic amino acid residues, usually in an amphipathic a helix (Fig 12-21).
• Table 1 Steps of protein import into mitochondria, from Neupert, 1997, Ann Rev Biochem, 66, 863-917.
  General import pathway
  1. Synthesis of proteins on cytosolic ribosomes as preproteins and release into the cytosol
  2. Transfer to the mitochondria assisted by cytosolic factors that help to maintain import competence and prevent aggregation.
  3.Recognition of preproteins by interaction of mitochondrial targeting signals with receptors on the surface of the outer membrane of mitochondria.
  4.Initiation of transfer through the preprotein translocase complex of the outer membrane (TOM complex). 5.Interaction of preproteins with the surface of the inner membrane and insertion into the preprotein translocase of the inner membrane (TIM complex) triggered by the membrane potential
  6. Completion of translocation through outer and inner membrane by the matrix-localized mt-Hsp70-ATP–dependent driving system associated with the TIM complex.
  7. Proteolytic processing of preproteins with cleavable targeting signals in the matrix.
  8. Folding of proteins in the matrix assisted by the molecular chaperone systems, mt-Hsp70, Hsp60, and associated co-chaperones.
  Specific targeting pathways
  1.Insertion of preproteins into the outer mitochondrial membrane facilitated by the TOM complex
  2. Translocation of preproteins across the outer membrane into the intermembrane space through the TOM complex. 3.Insertion of proteins into the inner membrane facilitated by both TOM and TIMcomplexes without passage through the matrix.
  4.Insertion of preproteins into the inner membrane after partial or complete passage through the matrix. 5.Translocation of preproteins into the intermembrane space after partial or complete passage through the matrix
  6. Translocation of cytochrome c across the outer membrane without mediation by the TOM complex

• Protein import into chloroplasts resembles mitochondrionís. Proteins are targeted by N-terminal sequences of 30-100 amino acids, called transit peptides, which direct translocation across the two membranes of chloroplasts and are then removed by proteolytic digestion. As in mitochondria, proteins are transported across the inner and outer chloroplast membranes at regions of close contacts between them. Also, translocation may require protein unfolding, so chaperones in the cytoplasmic and stromal side are involved, requiring energy in the form of ATP. Transit peptides are not positively charged and the translocation is not dependent on the electric potential across the membrane.
• Proteins transported into the thylakoid lumen are transported in two steps. They are first imported into the stroma as above, and are then translocated across the thylakoid membrane by a second hydrophobic signal sequence. This sequence will be cleaved by a peptidase in the thylakoid lumen.

5. Peroxisomes

• Surrounded by a single membrane, do not contain DNA or ribosomes.
• Contain oxidative proteins, such as catalase and urate oxidase. H₂O₂ produced in peroxisomes is harmful to cells, however, it is decomposed by catalase to water or used by the same enzyme to oxidize another organic compound (e.g. ethanol to acetaldehyde in liver and kidney).
• Peroxisomes are also involved in the synthesis of some lipids, i.e. cholesterol and dolichol, and in the breakdown of fatty acids by b oxidation.
• Once made in the cytosol, proteins are targeted to the interior of peroxisomes by at least two pathways. Most proteins are targeted by the simple Ser-Lys-Leu signal at their carboxy-terminus (peroxisome targeting signal 1, PTS1). Others are targeted by a sequence of nine amino acids (PST2) at their amino terminus. Some other PSTs may exist. Distinct internal signals are also responsible for targeting of integral membrane proteins to the peroxisome membrane. PTSs are not usually cleaved during the import of protein into the peroxisome.
• PST1 and PST2 are recognized by distinct receptors and transferred to a translocation complex that mediates their transfer through the organelleís membrane. Cytosolic Hsp70 has been implicated, but the possible role of molecular chaperones within the peroxisome lumen is unclear.