Synthesis of Membrane Proteins and the "Signal Hypothesis"

All of a cell’s proteins are synthesized by ribosomes, including, of course, those proteins that are destined for inclusion in the plasma membrane.

Cytoplasmic ri­bosomes in eukaryotic cells occur in two states: (1) “attached” (ribosomes associated with the mem­branes of the endoplasmic reticulum) and (2) “free” (ribosomes freely dispersed in the cytosol). Both attached and free ribosomes are be­lieved to contribute proteins to the plasma membrane.

Synthesis of Membrane Proteins and the “Signal Hypothesis”:

Principally as a result of the work of G. Blobel, D. D. Sabatini, C. M. Redman, C. Milstein, J. E. Rothman, J. Lenard, and H. F. Lodish, the mechanism that routes newly synthesized proteins to their proper des­tinations in the cell has gradually unfolded. A major contribution to this end has been the confirmation of the signal hypothesis proposed in the early 1970s by Blobel and Sabatini.

According to this hypothesis (Fig. 15-19), proteins that are to be either (a) secreted from the cell, (b) dispatched to lysosomes, or (c) incor­porated into the plasma membrane or membranes of the endoplasmic reticulum are encoded by mRNA molecules that contain a special nucleotide sequence called a “signal.”

The signal segment encodes a chain of about 16 to 26 amino acids that appears at or near the beginning of the polypeptide chain. Near its N- terminus, the signal sequence contains polar, basic residues, whereas the central domain is apolar. When a ribosome attaches to the mRNA in the cytosol and begins to translate the message, the signal sequence or signal peptide emerging from the ribosome is rec­ognized by a ribonucleoprotein complex in the cytosol called the signal recognition particle (i.e., SRP).

SRP, which consists of a 7 S cytoplasmic RNA mole­cule and six polypeptides, binds to the signal se­quence, bringing about a temporary halt to protein synthesis by that ribosome. Synthesis is resumed only if the SRP-ribosome complex attaches to the endo­plasmic reticulum; ribosomes synthesizing polypep­tides that lack a signal sequence do not interact with SRP and do not attach to the endoplasmic reticulum.

The amino acid sequences of a number of signal pep­tides have now been determined. Although they con­tain a specific distribution of hydrophobic and charged residues, no primary sequence homologies appear to exist. Consequently, it is be­lieved that SRP must recognize certain features con­tained in the signal peptide’s secondary and tertiary structure.

SRP-ribosome complexes attach to the endoplas­mic reticulum at specific sites occupied by SRP recep­tors (also called docking proteins). Once “docking” is completed, the SRP-ribosome-docking protein inter­action is replaced by a functional ribosome- membrane junction and the synthesis of the polypep­tide encoded by the mRNA is resumed. SRP returns to the cytosol where it can participate in another round of signal recognition and docking (i.e., the “SRP cycle” of Fig. 15-19a).

When protein synthesis is resumed by the docked ribosome, the elongating polypeptide chain passes through the ER membrane into the intracisternal space. This process, termed “translocation,” is presumed to involve active partici­pation of elements of the membrane. Translocation of the growing polypeptide into the intracisternal space is depicted in Figure 15-19 as taking place through a pore-like opening in the membrane solely to indicate that the membrane’s permeability barrier is tran­siently altered.

However, it is not known with cer­tainty whether translocation through the membrane takes place through such a proteinaceous water-filled channel or directly through the lipid bilayer. In most instances, the signal sequence is eventually cleaved from the remainder of the growing polypeptide by an extrinsic enzyme called signal peptidase attached to the lumenal surface of the ER.

If the protein being synthesized is destined for secretion from the cell, completion of synthesis is followed by the protein’s re­lease from the ribosome into the intracisternal space. The ribosome then detaches from the membrane and the mRNA and may participate in another round of protein synthesis. At the same time, the permeability barrier of the ER is restored.

Proteins discharged into the ER cisternae in this manner are ultimately con­veyed to the Golgi apparatus for chemical modifica­tion prior to secretion. In the case of pro­teins destined to be regular constituents of the endoplasmic reticulum or the plasma membrane, translocation into the intracisternal space is aborted before synthesis is finished so that the polypeptide is left anchored in the membrane (Fig. 15-19b). The in­formation halting the translocation is likely contained in the polypeptide itself and is recognized by the translocation apparatus.

For peripheral membrane proteins facing the exte­rior of the cell or the lumenal phase of the ER, the sig­nal sequence is followed by synthesis of the hydro- philic portion of the polypeptide. If an integral membrane protein is being synthesized, the hydro- philic portion is followed by a hydrophobic segment that remains anchored in the lipid bilayer. For pro­teins that span the membrane, synthesis is completed with the production of a final hydrophilic segment that faces the cytosol.

By comparing parts a and b of Figure 15-19, it is seen that the major distinction between the synthesis of secretory proteins and membrane proteins is that secretory proteins are released into the lumenal phase of the ER, whereas membrane proteins remain an­chored in the ER.

Addition of sugars to presumptive plasma membrane glycoproteins may occur soon after the hydrophilic portions of the molecules enter the ER cisternae. The membrane glycoprotein then migrates from the ER to the Golgi apparatus. Although the mechanism for this transfer is still uncertain, it is be­lieved to take place either by dispatchment of small vesicles from the ER, which then migrate to and fuse with the Golgi membranes, or by lat­eral “flow” along the ER membranes to the Golgi. Glycosylation of the membrane proteins is completed in the Golgi apparatus.

The Golgi apparatus dis­patches completed plasma membrane glycoproteins as small vesicles that migrate to and fuse with the plasma membrane. The overall process is summarized in Figure 15-20, which also shows that the intracellular/extracellular orientation of the mem­brane protein is maintained throughout its passage from the ER to the plasma membrane.

Some integral proteins have hydrophilic parts that face the interior of the cell and some peripheral proteins are associated only with the mem­brane’s cytoplasmic face. Although a similar mecha­nism is not precluded for the synthesis of these mem­brane proteins, they could be synthesized by free ribosomes.

Following release of these proteins in the cytosol they may diffuse to the plasma membrane. In­tegral proteins would be spontaneously inserted into the membrane by a hydrophobic segment (Fig. 15- 20), whereas peripheral proteins would attach to the membrane through polar interactions. Peripheral pro­teins reaching the plasma membrane in this manner could not pass through the membrane to the exterior surface because they could not traverse the hydropho­bic membrane core.

Prokaryotic cells do not contain an endoplasmic reticulum. Secretory proteins and new plasma membrane proteins are synthesized by ri­bosomes that attach to the inner surface of the plasma membrane. As in eukaryotic cells, ribosome attach­ment follows synthesis of a signal peptide encoded in the protein’s mRNA. The protein is then dispatched through the cell membrane and into the extracellular space.

Synthesis of Membrane Lipids:

In eukaryotic cells, phospholipid synthesis is associ­ated with the endoplasmic reticulum, whereas in pro­karyotic cells lipid synthesis is a property of the cyto­plasmic half of the plasma membrane. It is therefore likely that lipid synthesis in eukaryotic cells takes place in the cytoplasmic half of the ER membranes.

Following synthesis, the phospholipid becomes at least temporarily part of the interior monolayer but may ei­ther be enzymatically translocated to the outer layer or flip-flop between the two layers. In view of the fact that outer monolayer lipids are derived from the inner monolayer, an absolute asymmetry is precluded.

Because in prokaryotic cells lipid synthesis occurs in the plasma membrane, incorporation into the mem­brane’s structure is direct. In eukaryotic cells, how­ever, presumptive plasma membrane lipid must make its way from the ER to the plasma membrane.

This is believed to occur by one or both of two processes. Newly synthesized lipids inserted into ER membranes may make their way to the plasma membrane by the same mechanism that translocates ER membrane proteins (Fig. 15-20); that is both membrane proteins and lipids pass from the ER to the Golgi apparatus and are later dispatched to the plasma membrane via small vesicles.

The cytosol of eukaryotic cells contains a number of phospholipid transport proteins that function to transfer phospholipid molecules from one cellular membrane to another. These transport pro­teins might also play a major role in mediating the passage of phospholipids from the membranes of the ER to the plasma membrane.