Chapter 17: PROTEIN SORTING

Mitochondrial (MT) proteins:

∑ MT - structure (16-7); coding capacity (9-44); maternal inheritance; most proteins are encoded by the nuclear genome.

∑ MT protein uptake - experiment showing it occurs post-translationally (17-3); MT proteins are taken up at specific sites of inner-outer membrane contact as unfolded protein (17-5).

∑ MT targeting sequences - targeting sequence properties (T17-1); signals for targeting proteins to different MT compartments (17-2).

∑ MT import mechanism - Cytosolic ribosomes; Hsc70 chaperones; targeting sequence interaction with receptor; movement powered by proton motive force; matrix Hsc chaperones; removal of targeting sequence; folding into proper conformation (Hsc60) (17-4); targeting to MT inner membrane space (17-6); outer membrane proteins have matrix targeting and hydrophobic sequences that stop transfer; inner membrane proteins first move to the matrix and then get inserted into the membrane.

Chloroplast (CP) proteins:

∑ CP - structure; has own genome; most proteins are encoded by the nuclear genome.

∑ CP protein uptake - similarities to MT; targeting to the stroma (17-7); targeting to thalakoids (17-8).

Peroxisome protein import:

∑ Proteins synthesized in the cytoplasm; enter as folded proteins; requires ATP; import requires N or C terminal targeting sequences; catalase import mechanism (17-10).

Nuclear protein transport:

∑ Occurs through nuclear pores (11-28).

∑ Translocation of nuclear proteins - nuclear localization sequences (NLS); basic properties of NLSs and NESs; requirement for ATP; mechanism of transport to/from the cytoplasm (11-33, 11-37).

Protein secretion overview;

∑ Translated on the surface of the "rough" ER; demonstration that secreted proteins are initially located in the lumen of the ER (17-12); classes of secreted proteins (T17-3); pathway taken by secreted proteins (17-13); steps in the maturation of secreted proteins (17-14).

Synthesis of proteins at the ER membrane:

∑ Signal sequence (SS) - properties (T17-4); function; cleavage in ER by signal peptidase (17-15); N-terminal not Met.

∑ Signal recognition particle (SRP) - recognizes the SS; structure and function (17-17); binds to SRP receptor in the ER.

∑ Mechanism of protein translocation (17-16); the translocon is comprised of two proteins which form the channel through which the protein passes (17-18).

∑ Energetics of SRP binding to SS, SRP binding to SRP receptor and release of the SRP/receptor complex from the translocon (17-20).

Insertion of membrane proteins:

∑ Membrane protein orientation ≠ types (17-21); topogenic sequences; stop-transfer and membrane anchor sequence function (17-22); N_cytosol-C_luminal oriented proteins have an internal signal-anchor sequence (17-23); internal signal anchor sequences are also found on proteins inserted in the opposite orientation; for signal-anchor sequences, the flanking segment of signal-anchor sequences that carries the greatest positive charge will remain on the cytosolic face; multipass membrane proteins have multiple internal signal-anchor and stop-transfer membrane anchor sequences (17-24); certain transmembrane proteins having their N-terminus in the lumen are transferred to GPI anchors (17-25).

Post-translational modifications:

∑ Formation of disulfide bonds; allows for proper tertiary and quaternary structure; blocked in the cytosol by reduced glutathione, which is 50 fold more abundant than the oxidized form; many disulfide bonds form sequentially as the protein is translated; more complex disulfide bond arrangements are mediated by protein disulfide isomerase (PDI) in the ER lumen (17-26).

∑ Correct folding of proteins; performed in the ER; mediated by calnexin and calreticulin, which bind to carbohydrates (17-27) and peptidyl prolyl isomerases which accelerate rotation around prolyl bonds (cis ´ trans); proper folding enables appropriate contacts for the assembly of subunits; only properly folded proteins are transported to the Golgi; unfolded proteins trigger a response that results in greater synthesis of hsc70, calnexin, etc. (17-28); misfolded proteins are transported to the cytoplasm and degraded through the ubiquitin/proteasome pathway.

∑ Proteins that function in the ER are retrieved from the Golgi; the retrieval signal is a KDEL sequence at their C-termini; the KDEL binds to a receptor which shuttle proteins back to the ER in vesicles (17-29).

Protein Glycosylation:

∑ Most plasma membrane and secreted proteins are glcosylated and almost no cytoplasmic or nuclear proteins are glycosylated; glycosylation occurs in the ER; oligosaccahides can be O or N linked; O-linked occur at specific Ser and Thr residues and N-linked occur at Asn residues (17-30).

∑ O-linked glycosylation ≠ starts in ER and continues Golgi; sugar precursors are added by specific galactosyl transferases (17-32); ABO blood types (17-34, 17-35).

∑ N-linked glycosylation ≠ high mannose precursor synthesis on dolichol in the ER membrane (17-35); initiates in ER with addition of high mannose precursor to asparagine by oligosaccharide-protein transferase (17-36); processing of N-linked glycoproteins in different Golgi compartments (17-38); function to fold and stabilize glycoproteins.

∑ Targeting proteins to lysosomes ≠ phosphorylation of mannose on N-linked glycoproteins (17-39); GlcNAc phosphotransferase catalyzes the addition of phosphate to form M-6-P; targeting and recovery of lysosomal proteins by mannose-6 phosphate receptors in trans Golgi; receptor binds M-6-P tightly at ~pH7 and buds from trans-Golgi or PM; forms late endosome where protein is released and dephosphorylated(17-40); receptor is recycled.

Golgi/post-Golgi protein sorting and processing:

∑ Membrane spanning sequences cause retention of proteins in Golgi; different vesicles mediate constitutive and regulated secretion; proteins destined for regulated secretion form aggregates with chromogranin B and secretogranin II in low-pH trans Golgi; proteins not aggregated are secreted through the default continuous pathway.

∑ Proteolytic processing occurs in secretory vesicles; proproteins are cleaved by endoproteases at dibasic residues to generate the mature protein (17-42).

∑ Proteins in polarized cells are targeted to the apical and basolateral membranes; GPI anchors target proteins to the apical membrane; some proteins get targeted to the basolateral membrane by default and are then transported to the apical membrane via transcytosis (17-43).

Receptor mediated endocytosis:

∑ Pinocytosis ≠ unregulated intake of water from the extracellular space.

∑ Receptor mediated ≠ uses specific receptors on the PM and clathrin coated vesicles.

∑ Cholesterol intake ≠ LDL particle structure (17-45); mechanism of cholesterol intake (17-46).

∑ Iron intake ≠ mechanism (17-48).

∑ Maternal immunity ≠ transcytosis of immunoglobulins; driven by pH differences (17-49).

Vesicular transport mechanisms:

∑ Coated vesicles ≠ consist of COP I, COP II and clathrin; function in the formation/movement of vesicles between different compartments (17-50); adapter proteins select cargo for vesicles; GTP binding protein provides energy for vesicle formation (17-51).

∑ Clathrin coated vesicles ≠ structure (17-53); different adapter proteins mediate transport to different targets (AP2 = endocytosis; AP1 = Golgi to endosome; AP3 = Golgi to lysosome/vacuole); dynamin functions to pinch off the vesicle (17-54). the GTP-binding protein for clathrin is ARF; the clathrin coat is disassembled soon after vesicle formation by hsc70.

∑ COP I vesicles mediate retrograde transport within the Golgi and from Golgi to ER; coatomers form the coat around the vesicle (17-58); ARF is cytosolic and when bound to GTP binds to its receptor on the Golgi membrane; ARF binds to coatomers to promote vesicle formation along with fatty acyl-CoA.

∑ COP II vesicles mediate transport from ER to cis Golgi; Sar1 is the GTP binding protein used rather than ARF.

∑ V-SNARES are integral membrane proteins incorporated into vesicles that targets vesicles to the appropriate membrane (17-59); T-SNARES are found in the target membrane and bind to specific V-SNARES; SNAP25 and NSF mediate vesicle fusion; RAB proteins are GTP binding proteins that regulate the rate of vesicle fusion and are specific for certain vesicles.