chp 13 cell bio

• Mitochondrial and chloroplast proteins pass through the outer & inner membranes to enter the matrix or stromal space. Other proteins are sorted to other subcompartments of these organelles by additional sorting steps
• Nuclear proteins enter & exit through visible pores in the nuclear envelope
• Ribosomes synthesizing nascent proteins in the secretory pathway are directed to the rough endoplasmic reticulum (ER) by an ER signal seq
• After translation is completed on the ER these proteins can move via transport vesicles to the Golgi complex
• Schematic representation of protein synthesis on the ER. Note that membrane bound & free cytosolic ribosomes are identical. Membrane-bound ribosomes get recruited to the endoplasmic reticulum during protein synthesis of a polypeptide containing an ER signal sequence
• Cells are incubated w radiolabeled amino acids so that only newly synth proteins can become labeled
• The cells are then homogenized, fracturing the plasma membrane & shearing the rough ER into small vesicles called microsomes. Bc they have bound ribosomes, microsomes have much greater buoyant density than other membranous organelles and can be separated from them. --- purified microsomes.
• The purified microsomes r treated w protease in the presence or absence of a detergent
• The labeled secretory proteins associated w microsomes r digested by the protease only if the permeability barrier of the microsomal membrane is first destroyed by treatment with detergent. This finding indicates that the newly made proteins are inside the microsomes, equivalent to the lumen of the rough ER
• A hydrophobic N-terminal signal sequence targets Nascent secretory proteins to the ER
• A hydrophobic N-terminal signal sequence targets Nascent secretory proteins to the ER

  • N-terminus
  • Hydrophobic
  • 16- to 30-residues
  • Translation & translocation occur simultaneously
• Translation & translocation occur simultaneously. Cell-free experiment demonstrate that translocation of secretory proteins into microsomes is coupled to transition.
• Treatment of microsomes with EDTA which chelates Mg2+ ions strip them of associated ribosomes, allowing isolation of ribosome-free microsomes which r equivalent to ER membrane
• Proteins synthesis is carried out in a cell-free system containing functional ribosomes, tRNAs, ATP, GTP & cytosolic enzymes to which mRNA encoding a secretory protein is added
• The secretory protein is synthesized in the absence of microsomes (a) but is translocated across the vesicle membrane and loses its signal seq (decrease in molecular weight) only if microsomes r present during synthesis
• Cotranslational Translocation is initiated by the 2 GTP-Hydrolyzing proteins
• Chemical cross-linking experiments - in which amino acid side chains from a nascent secretory protein can become covalently attached to the Sec61alpha subunit- demonstrated that the translocating polypeptide chain comes into contact w the Sec61alpha protein, confirming its identity as the translocon pore
• 3 proteins called the Sec61 complex were found to form the mammalian translocon
• 3 proteins called the Sec61 complex : Sec61alpha, an integral membrane protein w 10 membrane-spanning helices and 2 smaller proteins, termed Sec61alpha and Sec61y
• ATP hydrolysis Power Post-translational Translocation of some secretory proteins in Yeast
• In most eukaryotes, secretory proteins enter the ER by cotranslational translocation.
• In yeast, some secretory proteins enter the ER lumen after translation has been completed. In such post-translational translocation, the translocating protein passes through the same Sec61 translocon that is used in cotranslational translocation
• In addition, the driving force for unidirectional translocation across the ER membrane is provided by an additional protein complex known as the Sec63 complex and a member of the Hsc 70 family of molecular chaperones known as BiP
• Post-translational translocation

  • Sec61 translocon
  • Sec63 complex
  • BiP
• The tetrameric Sec63 complex is embedded in the ER membrane in the vicinity of the translocon whereas BiP is within the ER lumen. Like other members of the Hsc70 family, BiP has a peptide-binding domain and an ATPase domain. These chaperones bind and stabilize unfolded or partially folded proteins
• post-translational translocation: interaction of the BiP*ATP w the luminal portion of the Sec63 complex causes hydrolysis of the bound ATP, producing a conformational change in BiP that promotes its binding to an exposed polypeptide chain
• Post-translational translocation : binding of a moL of BiP*ATP to the polypeptide prevents backsliding of the polypeptide out of the ER. As further inward random sliding exposes more of the polypeptide, successive binding of BiP*ADP moL to the polypeptide chain acts as a ratchet, ultimately drawing the entire polypeptide into the ER
• Post-translational translocation : on a slower time scale, the BiP moL spontaneously exchange their bound ADP for ATP, leading to release of the polypeptide which can then fold into its native conformation
• There r three main types of topogenic sequences that r used to direct proteins to the ER membrane and to orient them within it
• Internal Stop-transfer and signal-anchor sequences determine topology of single-pass proteins
• 3 main types of topogenic sequences:

  • N-terminal signal sequence
  • Internal sequences known as :

  Stop-transfer anchor sequence

  Signal-anchor sequence .
• a hydrophobic N-terminal signal sequence targets nascent secretory proteins to the ER
• Insertion of the Membrane proteins into the ER
• positioning type I single-pass proteins : the stop-transfer anchor sequence moves laterally between the translocon subunits and becomes anchored in the phospholipid bilayer. At this time. The translocon probably close
• positioning type I single-pass proteins: as synthesis continues, the elongating chain may loop out into the cytosol through the small space between the ribosome and translocon
• positioning type I single-pass proteins: when the synthesis is complete, the ribosomal subunits r released into the cytosol leaving the protein free to diffuse in the membrane
• When the seq of approximately 22 hydrophobic amino acids that will become a transmembrane domain of the nascent chain enters the translocon it stops transfer of the protein through the channel
• The Sec61 complex is then able to open like a clamshell allowing the hydrophobic transmembrane segment of the translocating peptide to move laterally btwn the protein domain constituting the translocon wall
• When the peptide exists the translocon in this manner, it becomes anchored in the phospholipid bilayer of the membrane
• in signal-anchor sequence, unlike Unlike type 1 proteins, type II & III proteins lack a cleavable N-terminal ER signal seq. instead, both possess a single internal hydrophobic signal-anchor seq that functions as both an ER signal sequence and a membrane-anchor sequence