Early Stages of the Secretory Pathway

In this section we take a closer look at vesicular traffic through the ER and Golgi stages of the secretory pathway i t-r-i

SNARE pair aire

COPII vesicle

Rough ER

COPII vesicle

Rough ER

Cis-

Golgi network

COPI vesicle

Coat protein

Cis-

Golgi network

COPI vesicle

Coat protein

J SNARE pair

cargo

Membrane receptor

Membrane cargo

▲ FIGURE 17-14 Vesicle-mediated protein trafficking between the ER and cis-Golgi. Steps 1 - 3: Forward (anterograde) transport is mediated by COPII vesicles, which are formed by polymerization of soluble COPII coat protein complexes (blue) on the ER membrane. v-SNAREs (red) and other cargo proteins (green) in the ER membrane are incorporated into the vesicle by interacting with coat proteins. Soluble cargo proteins (purple) are recruited by binding to appropriate receptors in the membrane of budding vesicles. Dissociation of the coat recycles free coat complexes and exposes v-SNARE proteins on the vesicle surface. After the uncoated vesicle becomes tethered to the cis-Golgi membrane in a Rab-mediated process, pairing between the exposed v-SNAREs and cognate t-SNAREs in the Golgi membrane allow vesicle fusion, releasing the contents into the cis-Golgi compartment (see Figure 17-11). Steps 4|- 6: Reverse (retrograde) transport, mediated by vesicles coated with COPI proteins (green), recycles the membrane bilayer and certain proteins, such as v-SNAREs and missorted ER-resident proteins (not shown), from the cis-Golgi to the ER. All SNARE proteins are shown in red although each v-SNARE and t-SNARE are distinct proteins.

vesicle transport serves to retrieve v-SNARE proteins and the membrane itself back to the ER to provide the necessary material for additional rounds of vesicle budding from the ER. COPI-mediated retrograde transport also retrieves missorted ER-resident proteins from the cis-Golgi to correct sorting mistakes. Proteins that have been correctly delivered to the Golgi advance through successive compartments of the Golgi by cisternal progression.

COPII Vesicles Mediate Transport from the ER to the Golgi

COPII vesicles were first recognized when cell-free extracts of yeast rough ER membranes were incubated with cytosol, ATP, and a nonhydrolyzable analog of GTP. The vesicles that formed from the ER membranes had a distinct coat, similar to that on COPI vesicles but composed of different proteins, designated COPII proteins. Yeast cells with mutations in the genes for COPII proteins are class B sec mutants and accumulate proteins in the rough ER (see Figure i7-5). Analysis of such mutants has revealed several proteins required for formation of COPII vesicles.

As described previously, formation of COPII vesicles is triggered when Seci2, a guanine nucleotide-exchange factor, catalyzes the exchange of bound GDP for GTP on Sari. This exchange induces binding of Sari to the ER membrane followed by binding of a complex of Sec23 and Sec24 proteins (see Figure i7-9). The resulting ternary complex formed between Sari- GTP, Sec23, and Sec24 is shown in Figure i7-i5. After this complex forms on the ER membrane, a second complex comprising Seci3 and Sec3i proteins then binds to complete the coat structure. A large fibrous protein, called Seci6, which is bound to the cytosolic surface of the ER, interacts with the Seci3/3i and Sec23/24 complexes, and acts to organize the other coat proteins, increasing the efficiency of coat polymerization.

Certain integral ER membrane proteins are specifically recruited into COPII vesicles for transport to the Golgi. The cytosolic segments of many of these proteins contain a di-acidic sorting signal (Asp-X-Glu, or DXE in the one-letter code). This sorting signal binds to the Sec24 subunit of the COPII coat and is essential for the selective export of certain membrane proteins from the ER (see Figure i7-i5). Biochemical and genetic studies currently are under way to identify additional signals that help direct membrane cargo proteins into COPII vesicles. Other ongoing studies seek to determine how soluble cargo proteins are selectively loaded into COPII vesicles. Although purified COPII vesicles from yeast cells have been found to contain a membrane protein that binds the soluble a mating factor, the receptors for other soluble cargo proteins such as invertase are not yet known.

The experiments described previously in which the transit of VSVG-GFP in cultured mammalian cells is followed by fluorescence microscopy (see Figure i7-2) provided insight into the intermediates in ER-to-Golgi transport. In some cells, small fluorescent vesicles containing VSVG-GFP could be seen to form from the ER, move less than i ^m, and

▲ FIGURE 17-15 Three-dimensional structure of ternary complex comprising the COPII coat proteins Sec23 and Sec24 and Sari ■ GTP. Early in the formation of the COPII coat, Sec23 (orange)/Sec24 (green) complexes are recruited to the ER membrane by Sari (red) in its GTP-bound state. In order to form a stable ternary complex in solution for structural studies, the nonhydrolyzable GTP analog GppNHp was used. A cargo protein in the ER membrane can be recruited to COPII vesicles by interaction of a tripeptide di-acidic signal (purple) in the cargo's cytosolic domain with Sec24. The likely position of the COPII vesicle membrane and the transmembrane segment of the cargo protein are indicated. The N-terminal segment of Sari that tethers it to the membrane is not shown. [See X. Bi et al., 2002, Nature 419:271; interaction with peptide courtesy of J. Goldberg.]

then fuse directly with the cis-Golgi. In other cells, in which the ER was located several micrometers from the Golgi complex, several ER-derived vesicles were seen to fuse with each other shortly after their formation, forming what is termed the "ER-to-Golgi intermediate compartment." These larger structures then were transported along microtubules to the cis-Golgi, much in the way vesicles in nerve cells are transported from the cell body, where they are formed, down the long axon to the axon terminus (Chapter 20). Microtubules function much as "railroad tracks" enabling these large aggregates of transport vesicles to move long distances to their cis-Golgi destination. At the time the ER-to-Golgi intermediate compartment is formed, some COPI vesicles bud off from it, recycling some proteins back to the ER.

COPI Vesicles Mediate Retrograde Transport within the Golgi and from the Golgi to the ER

COPI vesicles were first discovered when isolated Golgi fractions were incubated in a solution containing ATP, cytosol, and a nonhydrolyzable analog of GTP (see Figure i7-i0). Subsequent analysis of these vesicles showed that the coat is formed from large cytosolic complexes, called coatomers, composed of seven polypeptide subunits. Yeast cells containing temperature-sensitive mutations in COPI proteins accumulate proteins in the rough ER at the nonpermissive temperature and thus are categorized as class B sec mutants (see Figure 17-5). Although discovery of these mutants initially suggested that COPI vesicles mediate ER-to-Golgi transport, subsequent experiments showed that their main function is retrograde transport, both between Golgi cisternae and from the cis-Golgi to the rough ER (see Figure 17-14, right). Because COPI mutants cannot recycle key membrane proteins back to the rough ER, the ER gradually becomes depleted of ER proteins such as v-SNAREs necessary for COPII vesicle function. Eventually vesicle formation from the rough ER grinds to a halt; secretory proteins continue to be synthesized but accumulate in the ER, the defining characteristic of class B sec mutants.

As discussed in Chapter 16, the ER contains several soluble proteins dedicated to the folding and modification of newly synthesized secretory proteins. These include the chap-erone BiP and the enzyme protein disulfide isomerase, which are necessary for the ER to carry out its functions. Although such ER-resident luminal proteins are not specifically selected by COPII vesicles, their sheer abundance causes them to be continuously loaded passively into vesicles destined for the cis-Golgi. The transport of these soluble proteins back to the ER, mediated by COPI vesicles, prevents their eventual depletion

Most soluble ER-resident proteins carry a Lys-Asp-Glu-Leu (KDEL in the one-letter code) sequence at their C-terminus (see Table 17-2). Several experiments demonstrated that this KDEL sorting signal is both necessary and sufficient for retention in the ER. For instance, when a mutant protein disulfide isomerase lacking these four residues is synthesized in cultured fibroblasts, the protein is secreted. Moreover, if a protein that normally is secreted is altered so that it contains the KDEL signal at its C-terminus, the protein is retained in the ER. The KDEL sorting signal is recognized and bound by the KDEL receptor, a transmembrane protein found primarily on small transport vesicles shuttling between the ER and the cis-Golgi and on the cis-Golgi reticulum. In addition, soluble ER-resident proteins that carry the KDEL signal have oligosaccharide chains with modifications that are catalyzed by enzymes found only in the cis-Golgi or cis-Golgi reticulum; thus at some time these proteins must have left the ER and been transported at least as far as the cis-Golgi network. These findings indicate that the KDEL receptor acts mainly to retrieve soluble proteins containing the KDEL sorting signal that have escaped to the cis-Golgi network and return them to the ER (Figure 17-16).

The KDEL receptor and other membrane proteins that are transported back to the ER from the Golgi contain a Lys-Lys-X-X sequence at the very end of their C-terminal segment, which faces the cytosol (see Table 17-2). This KKXX sorting signal which binds to a complex of the COPI a and P subunits, is both necessary and sufficient to incorporate membrane proteins into COPI vesicles for retrograde transport to the ER. Temperature-sensitive yeast mutants lacking COPIa or COPIp not only are unable to bind the KKXX signal but also are unable to retrieve proteins bearing this signal back to the ER, indicating that COPI vesicles mediate retrograde Golgi-to-ER transport.

Clearly, the partitioning of proteins between the ER and Golgi complex is a highly selective and regulated process ultimately controlled by the specificity of cargo loading into both COPII (anterograde) and COPI (retrograde) vesicles. The selective entry of proteins into membrane-bounded transport vesicles, the recycling of membrane phospholipids and proteins, and the recycling of soluble luminal proteins between the two compartments are fundamental features of vesicular protein trafficking that also occur in later stages of the secretory pathway.

C/s-Golgi network

ER-to-Golgi transport vesicle

C/s-Golgi network

ER-to-Golgi transport vesicle

COPII coat

Rough ER

COPII coat

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