ABSTRACT

Many of the diverse functions of eukaryotic cells are carried out within discrete membrane-bound compartments or organelles that communicate with each other by membrane traffic pathways. My studies over the past few years, in collaboration with Richard Klausner and colleagues at NIH, have focused on the mechanisms by which cells maintain and remodel organelle structure and the role membrane traffic plays in this process. A key tool in our work has been the use of the fungal metabolite brefeldin A (BFA), which dramatically alters the distribution and flow of membrane through organelles. BFA reversibly blocks all forward, moving, or anterograde transport beyond the Golgi complex, causes the complete disassembly of Golgi stacks, and results in transport of Golgi components back to the endoplasmic reticulum (ER). These effects of BFA are not limited to Golgi membranes but extend to organelles of the endosomal system as well. Dissection of the biochemical basis of BFA's action has led to the finding that BFA blocks an essential activation step in the membrane interaction of a family of closely related proteins called ADP-ribosylation factors (ARFs), which are low-molecular-weight GTP-binding proteins distantly related to the ras proto-oncogene product. Blocking this activation step prevents assembly of a high-molecular-weight protein complex, called coatomer, onto the Golgi membrane. Because coatomer binding appears to be required both for the production of transport vesicles and for maintenance of Golgi structure, these findings suggest that dynamic processes of membrane traffic are integral to the existence and function of intracellular organelles.

QUESTIONS

Q: What was your starting point in this research, and how have your questions evolved?

A: The starting point in this research was the observation that although BFA efficiently blocks transport of proteins out of the ER, glycoproteins retained in the ER continue to be processed as if they were to be transported into the Golgi complex. A series of experiments employing diverse biochemical and morphological techniques showed that apparent maintenance of glycoprotein processing was due to redistribution of functional Golgi enzymes into the ER along a backward-moving, or retrograde, membrane-transport pathway. This finding prompted us to study the characteristics of this process, including how it occurred, its extent under normal conditions, and how BFA affected it. With this knowledge, we began using BFA as a new tool for investigating the biochemical basis for the regulation of membrane traffic and maintenance and the remodeling of organelle structure.

Q: Which findings have been most surprising to you or to other scientists?

A: As we were investigating the phenotypic effects of BFA and the biochemical basis of BFA action, we made several surprising findings. First, we found that redistribution of Golgi components into the ER during BFA treatment occurs through a unique retrograde membrane-transport pathway: Golgi-derived membrane is carried on long membrane tubules that migrate along microtubules to the cell periphery. This pathway is also important for recycling membrane back to the ER. Transport of membrane components along the retrograde pathway is normally selective, but in the presence of BFA, it becomes nonselective.

A second surprising finding is that the Golgi completely disassembles through the retrograde pathway during BFA treatment. This is the first example of a drug causing an organelle to disappear rapidly within cells, and it indicated that organelles, such as the Golgi, are not static, unchanging structures. The observation that the Golgi can reassemble equally rapidly into a functional complex upon BFA washout further supports our view of dynamic organelles and provides a novel approach to investigating organelle biogenesis.

The third surprise was our finding that upon BFA treatment a discrete population of peripheral membrane proteins [including coatomer and (ARF)] rapidly dissociate from Golgi membrane, before the Golgi membrane is redistributed into the ER. This observation indicated that BFA has a specific site of action on membrane, and this has set the stage for investigations of the biochemical basis for BFA effects and the regulation of membrane structure and traffic.

Q: What were the greatest stumbling blocks, and what new observations, techniques, reagents, or insights helped you get past them?

A: Our efforts at understanding the basis of BFA's effects on organelle structure and membrane traffic were not without stumbling blocks. We were quite fortunate, however, to have a wide variety of reagents to use in identifying organelles and membrane-transport intermediates in morphological studies. These were provided in large part by numerous very generous outside investigators. Also crucial for this work was our ability to adapt existing biochemical protocols and in vitro reconstitution assays to identify and characterize the peripheral membrane proteins whose association with Golgi membrane is affected by BFA.

Q: Do you see any potential areas where this research might provide insight to clinical scientists?

A: Our finding that the membrane traffic pathways and the very existence of some organelles can be reversibly regulated by a simple fungal metabolite, BFA, opens the possibility of selectively blocking intracellular traffic patterns by this class of drug. This offers tremendous therapeutic potential in the treatment of intracellular parasites and toxic and infectious agents, since virtually all these agents rely on host membrane traffic routes that are sensitive to BFA. Thus, for example, BFA can inhibit viral gene expression on the surface of cells and is, therefore, a potentially potent antiviral agent. In addition, BFA prevents various bacterial toxins, including botulinum toxin, from entering into cells. BFA can also block the presentation of antigens that initiate an immune response and thus may improve the course of autoimmune disease and graft rejection. Finally, BFA has been shown to have extraordinary selectivity in its effects on the growth of different populations of cells, suggesting potential as an anticancer agent. Studies with BFA, therefore, have refined our understanding of the control mechanisms underlying membrane traffic, thereby providing clinical and basic investigators with unparalleled power to manipulate the transport of molecules through the membrane systems of the cell.

Q: How are you following up on this work, and what questions would you ultimately like to answer?

A: My current work is focused on understanding how cells control the organization and distribution of organelles through membrane trafficking pathways and the role this plays in developmental and disease processes. For this, we are examining both simple eukaryotic cells including Toxoplasma gondii, where we hope to define the minimal requirements for secretory transport, and more complex cellular systems including mammalian tissues and embryos, where we hope to find new relationships between membrane organization and function. These studies will require an understanding of the role of microtubules, microtubule motor proteins, and cytosolic organelle-associated proteins in organelle localization and transport.