Endocytosis is a critical process that regulates nutrient uptake and cellular homeostasis, and is exploited for pathogen entry. The endocytic internalization process requires a precise sequence of events that begins with initiation and cargo selection and ends with scission and internalization of the endocytic vesicle. Numerous endocytic proteins and pathways have been identified;however, a key unanswered question is how endocytic proteins are recruited sequentially to fulfill their stage-specific functions. We previously discovered an endocytic function for the essential, yeast scaffold protein Pan1, a homologue of the mammalian endocytic protein Intersectin. Pan1 has conserved partner proteins that operate at the early coat (initiation), late coat (maturation) and final (scission/internalization) stages of endocytosis. For example, Pan1 binds Ede1, an early coat protein that acts during vesicle initiation. Pan1 also binds End3 and Sla2, two late coat proteins that act during vesicle maturation. Finally, Pan1 promotes invagination and scission through stimulating both Arp2/3 and the type I myosins Myo3/5 that mediate actin polymerization at the final stage of vesicle invagination/scission. Thus, we propose that Pan1 contributes to the sequential recruitment of endocytic factors, thereby regulating transitions from (1) initiation to maturation, and (2) maturation to final endocytic stages. We also discovered a new early coat protein, Syp1, which forms an early coat complex with Ede1 and inhibits Las17/WASp-dependent Arp2/3 actin polymerization. We propose that Syp1 cargo-binding and membrane tabulation activities contribute to regulation transitions between endocytic stages. Knowing the mechanisms that regulate the endocytic machinery is a prerequisite for a complete understanding of the fundamental process of endocytosis. This information may also help explain the pathology of numerous human diseases, entry routes of pathogens such as viruses or bacteria, and how drug delivery and gene therapy reagents gain access to cells, all of which has high relevance to human health.

Public Health Relevance

Many components of the endocytic machinery that we study are conserved in yeast and humans, and have been implicated in diseases, including Alzheimer's disease (PICALM/Yap180s), Huntington's Disease (Hip1R/Sla2), Liddle's Syndrome (Nedd4/Rsp5), Wiskott-Aldrich Syndrome (WASp/Las17), and numerous leukemias that arise due to chromosomal translocations that create chimeric fusions with endocytic proteins. Better understanding of the functions of these proteins will lead to insight into the pathological mechanisms of these diseases. In addition, some viruses and bacteria toxins enter cells via endocytosis. Gene therapy and other therapeutic strategies rely upon endocytosis for delivery of the treatment to the cytoplasm of appropriate cells. Thus, a better appreciation of the function and regulation of the components of endocytic pathways may ultimately facilitate the design of better treatment strategies for a wide range of human ailments.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060979-13
Application #
8485618
Study Section
Special Emphasis Panel (ZRG1-CB-B (02))
Program Officer
Ainsztein, Alexandra M
Project Start
2000-03-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
13
Fiscal Year
2013
Total Cost
$402,807
Indirect Cost
$152,826
Name
Johns Hopkins University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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