[Abstract unavailable

Longstanding evidence supports a role for actin in endocytosis; an intact actin cytoskeleton is required for endocytosis in yeast, and drugs that inhibit actin polymerization inhibit endocytosis in both yeast and mammalian cells. The yeast Arp2/3 complex is required for the internalization step of endocytosis. In addition, some early endocytic events in mammalian cells are associated with the formation of actin tails similar to those generated by activated Arp2/3 complex. However, until now no Arp2/3 complex activator has been identified among proteins known to mediate early steps in endocytosis. Here we show that the yeast endocytic protein Pan1p binds to and activates the Arp2/3 complex. Genetic interactions between PAN1 and mutants of Arp2/3 subunits, or of the Arp2/3 activator LAS17, provide evidence for this activity in vivo. We suggest that Pan1p forms the core of an endocytic complex and physically couples actin polymerization nucleated by the Arp2/3 complex to the endocytic machinery, thus providing the forces necessary for endocytosis.

Clathrin-coated vesicles (CCVs) are a central component of endocytosis and traffic between the trans-Golgi network (TGN) and endosomes. Although endocytic CCV formation is well characterized, much less is known about CCV formation at internal membranes. Here we describe two epsin amino-terminal homology (ENTH) domain-containing proteins, Ent3p and Ent5p, that are intimately involved in clathrin function at the Golgi. Both proteins associate with the clathrin adaptor Gga2p in vivo; Ent5p also interacts with the clathrin adaptor complex AP-1 and clathrin. A novel, conserved motif that mediates the interaction of Ent3p and Ent5p with gamma-ear domains of Gga2p and AP-1 is defined. Ent3p and Ent5p colocalize with clathrin, and cells lacking both Ent proteins exhibit defects in clathrin localization and traffic between the Golgi and endosomes. The findings suggest that Ent3p and Ent5p constitute a functionally related pair that co-operate with Gga proteins and AP-1 to recruit clathrin and promote formation of clathrin coats at the Golgi/endosomes. On the basis of our results and the established roles of epsin and epsin-related proteins in endocytosis, we propose that ENTH-domain-containing proteins are a universal component of CCV formation.

With the advent of complete genome sequences, large-scale functional analyses are generating new excitement in biology and medicine. To facilitate genomewide functional analyses, we developed a high-density cell array with quantitative and automated readout of cell fitness. Able to print at >×10 higher density on a standard microtiter plate area than currently possible, our cell array allows single-plate screening of the complete set of Saccharomyces cerevisiae gene-deletion library and significantly reduces the amount of small molecules and other materials needed for the study. We used this method to map the relation between genes and cell fitness in response to rapamycin, a medically important natural product that targets the eukaryotic kinase Tor. We discuss the implications for pharmacogenomics and the uncharted complexity in genotype-dependent drug response in molecularly targeted therapies. Our analysis leads to several basic findings, including a class of gene deletions that confer better fitness in the presence of rapamycin. This result provides insights into possible therapeutic uses of rapamycin/CCI-779 in the treatment of neurodegenerative diseases (including Alzheimer's, Parkinson's, and Huntington's diseases), and cautions the possible existence of similar rapamycin-enhanceable mutations in cancer. It is well established in yeast that although TOR2 has a unique rapamycin-insensitive function, TOR1 and TOR2 are interchangeable in the rapamycin-sensitive functions. We show that even the rapamycin-sensitive functions are distinct between TOR1 and TOR2 and map the functional difference to a ≈120-aa region at the N termini of the proteins. Finally, we discuss using cell-based genomic pattern recognition in designing electronic or optical biosensors.

Background: During energy starvation, cells utilize intracellular resources for survival; however, the resources used during glucose starvation are unknown. Results: In glucose-starved yeast, vacuolar hydrolysis and endocytosis promote survival whereas autophagy is dispensable. Conclusion: Vacuolar hydrolysis blocks autophagy and provides resources for survival during glucose starvation. Significance: This new survival mechanism could protect cells from starvation in many situations.