Métodos de diseño y el código de AISC [7]

The translocation across the ER membrane has been

reviewed by Walter and Lingappa (1986) and, more recently, by Pugsley (1990). Walter and Lingappa suggest that the two questions to be addressed when considering ER translocation are:

(i) how is the target membrane recognised, and

(ii) once targeted to the membrane surface, how is the protein translocated across the membrane?

(1) Recognition of Target membrane.

Proteins destined for the secretory pathway are

distinguished by the presence of signal peptides. These signals are discrete N-terminal extensions that are cleaved from the

mature protein during translocation (for review of signal

peptides see von Heijne, 1990). They have very little or no amino acid (primary) sequence homology but comparison of many known signal peptides has elucidated three functional domains: a positively-charged amino-terminus, or n-region; a hydrophobic h-region; and a carboxy-terminal c-region which contains polar residues and contains the signal peptide cleavage site (von

Wiech et aj. (1990) have recently collated these theories and define a translocation competent state as meaning that the

protein must be water-soluble, possess secondary but no

tertiary structure, and interact with cytosolic 'chaperones'. This last term has been used to describe proteins that affect the folding of others without playing any part in their final

structure (see Ellis and Hemmingsen, 1989). The signal

recognition particle (see below) and members of the heat shock protein family (hsp70) (Chirico et al, 1988) act as such chaperones in the translocation of proteins across the ER lumen.

1.3.3 TRANSLOCATION ACROSS THE ER MEMBRANE

The translocation across the ER membrane has been

reviewed by Walter and Lingappa (1986) and, more recently, by Pugsley (1990). Walter and Lingappa suggest that the two questions to be addressed when considering ER translocation are:

(i) how is the target membrane recognised, and

(ii) once targeted to the membrane surface, how is the protein translocated across the membrane?

(i) Recognition of Target membrane.

Proteins destined for the secretory pathway are

distinguished by the presence of signal peptides. These signals are discrete N-terminal extensions that are cleaved from the mature protein during translocation (for review of signal peptides see von Heijne, 1990). They have very little or no amino acid (primary) sequence homology but comparison of many known signal peptides has elucidated three functional domains: a positively-charged amino-terminus, or n-region; a hydrophobic h-region; and a carboxy-terminal c-region which contains polar residues and contains the signal peptide cleavage site (von

Heijne, 1985). The n- and h-regions are known to be involved in membrane targeting - the hydrophobic core forming an alpha

helix. The c-region often contains alpha-helix breaking

residues such as glycine and proline, and residues at positions -3 and -1 relative to the cleavage site tend to be small and

uncharged. All plant storage proteins synthesised as

preproproteins carry N-terminal extensions that fit this

description.

The signal peptide interacts with a cytosolic factor,

the signal recognition particle (SRP). SRP, an 11S small

cytoplasmic ribonucleoprotein, was purified by Walter and

Blobel (1980) and consists of a 7S RNA component and 6 polypeptide chains. During protein synthesis, SRP recognises the signal peptide as it emerges from the ribosome, binds to the nascent chain and arrests further polypeptide elongation. Krieg et a_i (1986) showed that the 54kD monomeric polypeptide of SRP contained the signal peptide binding site. Sequencing of cDNA encoding the 54kD protein (Bernstein et al, 1989: Romish et al, 1989) has revealed a methionine-rich binding pocket which may accommodate the hydrophobic signal sequence. The deduced amino acid sequence also reveals a potential 6TP

binding site. Pugsley (1990) suggests that GTP

binding/hydrolysis at this site may affect the affinity of SRP for the signal peptide.

The SRP/nascent polypeptide/ribosome complex then

interacts with another characterised protein factor, the SRP receptor (Walter and Blobel, 1981) or docking protein (Meyer et a l . 1982). This is a heterodimeric integral membrane protein consisting of a 69kD trans-membrane alpha chain and a 30kD beta chain. A hydrophilic cytosolic domain of the alpha subunit strongly resembles a nucleotide binding site and Walter and Lingappa (1986) suggest that the RNA component of SRP may interact with this domain.

At this stage the ribosome binds to the membrane in a functional way. Meyer's group have recently purified a 180 kD integral ER membrane protein which binds to ribosomes and that they propose is the ribosome binding factor (unpublished).

The next step in the translocation process is the GTP- mediated release of the SRP from the signal peptide (Connolly and Gilmore, 1989). This allows the recycling of SRP, its receptor and the resumption of peptide elongation. A second membrane protein, identified by Weidmann e_t ¿1 (1987) and termed the Signal Sequence Receptor (SSR), (see also Walter, 1987) binds the signal peptide and may be involved in the release of the SRP from the signal peptide (reviewed by Rapoport, 1990).

(ii) Translocation of the nascent peptide.

Although signal peptides are hydrophobic and can insert into lipid monolayers (Cornell et al, 1989), the mature portion of the protein is unlikely to pass through the lipid bilayer. Instead, integral membrane proteins are thought to assemble to form a channel, or translocon, through which the protein is translocated (see Pugsley, 1990). The SSR mentioned above may form part of this translocon (Rapoport, 1990).

Plant storage preproprotein precursors can be

translocated across mammalian membranes and the involvement of the SRP in this translocation has been demonstrated using globulin precursors from Vicia faba (Bassuner e£ al, 1984). This suggests that similar mechanisms are involved in the targeting to and translocation across the plant cell ER membrane although there is no direct data on the plant components themselves.