October 19, 2012

Anchor sequences determine protein orientation

  • An anchor (stop-transfer) (often referred to as a "transmembrane anchor") is a segment of a transmembrane protein which resides in the membrane.
  • An anchor sequence halts the passage of a protein through the translocon. Typically this is located at the C-terminal end and results in a group I orientation in which the N-terminus has passed through the membrane.
  • A combined signal-anchor sequence can be used to insert a protein into the membrane and anchor the site of insertion. Typically this is internal and results in a group II orientation in which the N-terminus is cytosolic. 

October 18, 2012

Proteins reside in membranes by means of hydrophobic regions

  • The transmembrane region (transmembrane domain) is the part of a protein that spans the membrane bilayer. It is hydrophobic and in many cases contains approximately 20 amino acids that form an α-helix. It is also called the transmembrane domain.
  • A transmembrane protein (Integral membrane protein) extends across a lipid bilayer. A hydrophobic region (typically consisting of a stretch of 20-25 hydrophobic and/or uncharged aminoa acids) or regions of the protein resides in the membrane. Hydrophilic regions are exposed on one or both sides of the membrane.

Reverse translocation sends proteins to the cytosol for degradation

  • Retrograde translocation (Reverse translocation) is the translocation of a protein from the lumen of the ER to the cytoplasm. It usually occurs to allow misfolded or damaged proteins to be degraded by the proteasome.
  • Sec61 translocons can be used for reverse translocation of proteins from the ER into the cytosol. 

Translocation requires insertion into the translocon and (sometimes) a ratchet in the ER

  • The ribosome, SRP, and SRP receptor are sufficient to insert a nascent protein into a translocon.
  • Proteins that are inserted post-translationally require additional components in the cytosol and Bip in the ER.
  • Bip is a ratchet that prevents a protein from slipping backward.
The translocon and the SRP receptor are the basic components required for co-translational translocation. When the Sec61 complex is incorporated into artificial membranes together with the SRP receptor, it can support translocation of some nascent proteins. Other nascent proteins require the presence of an additional component, TRAM, which is a major protein that becomes crosslinked to a translocating nascent chain. TRAM stimulates the translocation of all proteins.

The translocon forms a pore

  • A translocon is a discrete structure in a membrane that forms a channel through which (hydrophilic) proteins may pass.
  • The Sec61 trimeric complex provides the channel for proteins to pass through a membrane.
  • A translocating protein passes directly from the ribosome to the translocon without exposure to the cytosol. 

The SRP interacts with the SRP receptor

  • The Alu domain comprises the parts of the 7S RNA of the SRP that are related to Alu RNA.
  • The S domain is the sequence of 7S RNA of the SRP that is not related to Alu RNA.
  • The SRP is a complex of 7S RNA with 6 proteins.
  • The bacterial equivalent to the SRP is a complex of 4.5S RNA with two proteins.
  • The SRP receptor is a dimer.
  • GTP hydrolysis releases the SRP from the SRP receptor after their interaction. 

The signal sequence interacts with the SRP

  • Protein translocation describes the movement of a protein across a membrane. This occurs across the membranes of organelles in eukaryotes, or across the plasma membrane in bacteria. Each membrane across which proteins are translocated has a channel specialized for the purpose.
  • The signal recognition particle (SRP) is a ribonucleoprotein complex that recognizes signal sequences during translation and guides the ribosome to the translocation channel. SRPs from different organisms may have different compositions, but all contain related proteins and RNAs.
  • Signal peptidase is an enzyme within the membrane of the ER that specifically removes the signal sequences from proteins as they are translocated. Analogous activities are present in bacteria, archaebacteria, and in each organelle in a eukaryotic cell into which proteins are targeted and translocated by means of removable targeting sequences. Signal peptidase is one component of a larger protein complex.

Signal sequences initiate translocation

  • Protein sorting (targeting) is the direction of different types of proteins for transport into or between specific organelles.
  • A signal sequence is a short region of a protein that directs it to the endoplasmic reticulum for co-translational translocation.
  • Proteins associate with the ER system only co-translationally.
  • The signal sequence of the substrate protein is responsible for membrane association. 

Proteins that associate with membranes via N-terminal leaders use a hierarchy of signals to find their final destination. In the case of the reticuloendothelial system, the ultimate location of a protein depends on how it is directed as it transits the endoplasmic reticulum and Golgi apparatus. The leader sequence itself introduces the protein to the membrane; the intrinsic consequence of the interaction is for the protein to pass through the membrane into the compartment on the other side. For a protein to reside within the membrane, a further signal is required to stop passage through the membrane. Other types of signals are required for a protein to be sorted to a particular destination, that is, to remain within the membrane or lumen of some particular compartment. The general process of finding its ultimate destination by transport through successive membrane systems is called protein sorting or targeting, and is discussed in 27 Protein trafficking.

The overall nature of the pathway is summarized in Figure 8.18. The "default pathway" takes a protein through the ER, into the Golgi, and on to the plasma membrane. Proteins that reside in the ER possess a C-terminal tetrapeptide (KDEL, which actually provides a signal for them to return to the ER from the Golgi). The signal that diverts a protein to the lysosome is a covalent modification: the addition of a particular sugar residue. Other signals are required for a protein to become a permanent constituent of the Golgi or the plasma membrane. We discuss direction to these locations in 27 Protein trafficking.

There is a common starting point for proteins that associate with, or pass through, the reticuloendothelial system of membranes. These proteins can associate with the membrane only while they are being synthesized. The ribosomes synthesizing these proteins become associated with the endoplasmic reticulum, enabling the nascent protein to be co-translationally transferred to the membrane. Regions in which ribosomes are associated with the ER are sometimes called the "rough ER," in contrast with the "smooth ER" regions that lack associated polysomes and which have a tubular rather than sheet-like appearance (for review see Palade, 1975). Figure 8.19 shows ribosomes in the act of transferring nascent proteins to ER membranes.
The proteins synthesized at the rough endoplasmic reticulum pass from the ribosome directly to the membrane. Then they are transferred to the Golgi apparatus, and finally are directed to their ultimate destination, such as the lysosome or secretory vesicle or plasma membrane. The process occurs within a membranous environment as the proteins are carried between organelles in small membrane-coated vesicles (see 27 Protein trafficking.)

Co-translational insertion is directed by a signal sequence. Usually this is a cleavable leader sequence of 15-30 N-terminal amino acids. At or close to the N-terminus are several polar residues, and within the leader is a hydrophobic core consisting exclusively or very largely of hydrophobic amino acids (for review see von Heijne, 1985). There is no other conservation of sequence. Figure 8.20 gives an example.
The signal sequence is both necessary and sufficient to sponsor transfer of any attached polypeptide into the target membrane. A signal sequence added to the N-terminus of a globin protein, for example, causes it to be secreted through cellular membranes instead of remaining in the cytosol (Lingappa et al., 1984).
The signal sequence provides the connection that enables the ribosomes to attach to the membrane. There is no intrinsic difference between free ribosomes (synthesizing proteins in the cytosol) and ribosomes that are attached to the ER. A ribosome starts synthesis of a protein without knowing whether the protein will be synthesized in the cytosol or transferred to a membrane. It is the synthesis of a signal sequence that causes the ribosome to associate with a membrane (Blobel and Dobberstein, 1975; for review see Lee and Beckwith, 1986).

October 16, 2012

Hsp60/GroEL forms an oligomeric ring structure

  • Hsp60/GroEL forms an oligomeric structure consisting of 14 subunits arranged in two inverted heptameric rings.
  • A GroES heptamer forms a dome that caps one end of the double ring.
  • A substrate protein undergoes a cycle of folding in the cavity of one of the Hsp60/GroEL rings. It is released and rebound for further cycles until it reaches mature conformation.
  • Hydrolysis of ATP provides energy for the folding cycles.

The Hsp70 family is ubiquitous

  • Hsp70 is a chaperone that functions on target proteins in conjunction with DnaJ and GrpE.
  • Members of the Hsp70 family are found in the cytosol, in the ER, and in mitochondria and chloroplasts.
The Hsp70 family is found in bacteria, eukaryotic cytosol, in the endoplasmic reticulum, and in chloroplasts and mitochondria (for review see Frydman, 2001). A typical Hsp70 has two domains: the N-terminal domain is an ATPase; and the C-terminal domain binds the substrate polypeptide (Flaherty, DeLuca-Flaherty, and McKay, 1990; Zhu et al., 1996). When bound to ATP, Hsp70 binds and releases substrates rapidly; when bound to ADP, the reactions are slow. Recycling between these states is regulated by two other proteins, Hsp40 (DnaJ) and GrpE.