RME-8

 

---

In order to identify endocytic genes essential to survival, we employed a temperature sensitive (ts) screen to isolate mutants defective in YP170::GFP endocytosis. In this ts screen we isolated rme-8 (b1023ts). rme-8 (b1023) is a recessive ts lethal mutation. rme-8(b1023) worms are viable at 15°C, which is their permissive temperature, but YP170::GFP uptake by oocytes is strongly blocked even at this temperature. When examined by confocal microscopy, the number of yolk platelets in rme-8 (b1023) oocytes is greatly reduced and the yolk platelets appear to be half as large as those in wild type oocytes. rme-8(b1023) is also ts-defective in fluid-phase endocytosis. Temperature-shift experiments indicate a continuous requirement for rme-8 activity during development. Adults shifted to the non-permissive temperature produce only dead embryos. Larvae shifted to the non-permissive temperature die during the next molt, indicating a role for RME-8 mediated endocytosis in molting. We found that rme-8 encodes a large predicted protein (2279 amino acids) with a central DnaJ domain and four novel IWN domains. Close homologues of rme-8 are found in the Arabidopsis genomic sequence database and human and Drosophila ESTs, but not in the yeast genome. The amino acid sequence identity among these homologues extends throughout the predicted protein sequence. There is 42% identity and 61% similarity with a human cDNA in the C-terminal 1047 aa. C. elegans rme-8 is the first of these genes to be studied functionally. We are taking several approaches to understand the function of RME-8 in endocytosis. We are examing the expression pattern and subcellular localization of RME-8 using an RME-8::GFP reporter and anti-RME-8 antibodies. We are analyzing the function of RME-8's domains by creating deletions and assaying their in vivo functions and localizations. We will also identify RME-8 interacting proteins with the yeast two-hybrid system. Together, these experiments will establish RME-8's role in a specific step in endocytosis and its possible role in a protein complex. Understanding RME-8 in C. elegans will provide an important basis for understanding the functions of its close homologues in higher organisms, particularly humans.

---

Back to Home