As we have seen in this chapter, we now understand many aspects of the basic processes responsible for selectively transporting proteins into the endoplasmic reticulum (ER), mitochondrion, chloroplast, peroxisome, and nucleus. Biochemical and genetic studies, for instance, have identified sequences responsible for targeting proteins to the correct organelle membrane and the membrane receptors that recognize these targeting sequences. We have also learned much about the underlying mechanisms that translocate proteins across organelle membranes, and we have determined whether energy is used to push or pull proteins across the membrane in one direction, the types of channels through which proteins pass, and whether proteins are translocated in a folded or an unfolded state. Nonetheless, fundamental questions remain unanswered; probably the most puzzling is how fully folded proteins move across a membrane.

The peroxisomal import machinery provides one example of the translocation of folded proteins. It not only is capable of translocating fully folded proteins with bound cofactors into the peroxisomal matrix, it can even direct the import of a large gold particle decorated with a peroxisomal targeting peptide. Some researchers have speculated that the mechanism of peroxisomal import may be related to that of nuclear import, the best-understood example of post-translational translocation of folded proteins. Both the peroxisomal and nuclear import machinery can transport folded molecules of very divergent sizes, and both appear to involve a component that cycles between the cytosol and the organelle interior—the Pex5 PTS1 receptor in the case of peroxisomal import and the Ran-importin complex in the case of nuclear import. However, there also appear to be crucial differences between the two translocation processes. For example, nuclear pores are large, stable macromolecular assemblies that are readily observed by electron microscopy, whereas analogous porelike structures have not been observed in the peroxisomal membrane. Moreover, small molecules can readily pass through nuclear pores, whereas peroxisomal membranes maintain a permanent barrier to the diffusion of small hydrophilic molecules. Taken together, these observations suggest that peroxisomal import may require an entirely new type of translocation mechanism.

The evolutionarily conserved mechanisms for translocating folded proteins across the plasma membrane of bacterial cells and across the thylakoid membrane of chloroplasts are also poorly understood. A better understanding of all these processes for translocating folded proteins across a membrane will probably hinge on future development of in vitro translocation systems that allow investigators to define the biochemical mechanisms driving translocation and to identify the structures of trapped translocation intermediates.

Compared with our understanding of how soluble proteins are translocated into the ER lumen and mitochondrial matrix, our understanding of how targeting sequences specify the topology of multipass membrane proteins is quite elementary. For instance, we do not know how the translocon channel accommodates polypeptides that are oriented differently with respect to the membrane, nor do we understand how local polypeptide sequences interact with the translocon either to set the orientation of transmembrane spans or to signal for lateral passage into the membrane bilayer. A better understanding of how the amino acid sequences of membrane proteins can specify membrane topology will be crucial for decoding the vast amount of structural information for membrane proteins contained within databases of genomic sequences.

A more detailed understanding of all translocation processes should continue to emerge from genetic and biochemical studies, both in yeasts and in mammals. These studies will undoubtedly reveal additional key proteins involved in the recognition of targeting sequences and in the translocation of proteins across lipid bilayers. Finally, the structural studies of translocons will probably be extended in the future to reveal, at resolutions on the atomic scale, the conformational states that are associated with each step of the translocation cycle.