J. Andrew Whitney

Cytoplasmic coat proteins involved in endosome function

Endosomes are intermediates for a complex series of sorting and transport events that occur during receptor-mediated endocytosis. These involve the recognition of targeting determinants on the cytoplasmic domains of many membrane proteins as well as the formations of specific transport vesicles. Accordingly, endosome function is likely to be governed by the regulated assembly of cytoplasmic coat complexes. We have found that, in vitro, endosomes recruit a characteristic set of cytoplasmic proteins in a GTP gamma S-stimulated and brefeldin A-sensitive fashion. Among these are members of the COP-I and ARF families of coat proteins. In addition, endosomes were also found to assemble distinct, clathrin-like coats. Since microinjection of antibodies to beta-COP inhibits the entry of enveloped viruses via the endocytic pathway, it is apparent that the recruitment of COP-I or COP-I-related proteins plays an important role in the function of endosomes in intact cells.

Selective inhibition of transcytosis by brefeldin A in MDCK cells

Treatment of most cells with brefeldin A (BFA) leads to the retrieval of the Golgi complex to the endoplasmic reticulum, presumably reflecting an inhibition of cytoplasmic coat protein binding to Golgi membranes. Although BFA has been thought to act only on biosynthetic organelles, we now show that this drug also reversibly blocks polymeric immunoglobulin receptor-mediated transcytosis in MDCK cells. The action of BFA on transcytosis was selective, since internalization, recycling, and intracellular degradation were unaffected. The block occurred early on the transcytotic pathway, probably before the translocation of IgA-containing vesicles from the basal to the apical cytoplasm. Although BFA caused MDCK cell endosomes to become more tubular, the organization of the Golgi and binding of the 110 kd Golgi coat protein beta-COP was surprisingly unaffected. These results suggest that in MDCK cells, endocytic organelles contain a BFA-sensitive coat that regulates their organization and function even though the Golgi coat is BFA resistant.

Brefeldin A and the endocytic pathway. Possible implications for membrane traffic and sorting.

A number of recent observations have suggested that the endocytic and biosynthetic pathways may share fundamentally similar transport mechanisms at the molecular level. Some of the more striking of these suggestions have come from a comparison of the effects of the macrocyclic lactone brefeldin A (BFA) on endosomes and the Golgi complex. BFA is thought to affect Golgi‐specific coat proteins that may be involved in maintaining the structural integrity of the organelle and in regulating membrane transport in the secretory pathway. Many of the effects of BFA on the endocytic system, such as the guanine nucleotide and aluminum fluoride (AlF− 4)‐regulated induction of microtubule‐dependent endosomal tubules, are strikingly reminiscent of the action of the drug on the Golgi complex. Therefore, the similar mechanisms of action of the drug on endosomes suggest that organelles of the endocytic pathway may be associated with similar cytoplasmic coats that could regulate endosome function and integrity.

Cloning, expression, and localization of a novel γ-adaptin-like molecule

We describe the cloning, expression, and localization of γ2‐adaptin, a novel isoform of γ‐adaptin. The predicted human and mouse γ2‐adaptin proteins are ∼90 kDa and 64.4% and 61.7% identical to γ‐adaptin, respectively. γ2‐Adaptin was localized to the Golgi, its localization distinct from γ‐adaptin. The membrane association of γ‐ and γ2‐adaptin could further be distinguished by differential sensitivity to the fungal metabolite brefeldin A, γ2‐adaptin binding being insensitive to drug treatment. Together, these results suggest that γ2‐adaptin plays a role in membrane transport distinct from that played by γ‐adaptin.