Abstract
As part of a high-throughput subcellular localisation project, the protein encoded by the RIKEN mouse cDNA 2610528J11 was expressed and identified to be associated with both endosomes and the plasma membrane. Based on this, we have assigned the name TEMP for Type III Endosome Membrane Protein. TEMP encodes a short protein of 111 amino acids with a single, alpha-helical transmembrane domain. Experimental analysis of its membrane topology demonstrated it is a Type III membrane protein with the amino-terminus in the lumenal, or extracellular region, and the carboxy-terminus in the cytoplasm. In addition to the plasma membrane TEMP was localized to Rab5 positive early endosomes, Rab5/Rab11 positive recycling endosomes but not Rab7 positive late endosomes. Video microscopy in living cells confirmed TEMP’s plasma membrane localization and identified the intracellular endosome compartments to be tubulovesicular. Overexpression of TEMP resulted in the early/recycling endosomes clustering at the cell periphery that was dependent on the presence of intact microtubules. The cellular function of TEMP cannot be inferred based on bioinformatics comparison, but its cellular distribution between early/recycling endosomes and the plasma membrane suggests a role in membrane transport.
Highlights
Determination of the subcellular distribution of individual proteins is essential to understanding a proteins function
TEMP clearly co-localises with sorting nexin 1 (SNX1) positive structures (Figure 4B±D) demonstrating that it is present on early endosomes
Truncation of the carboxy-terminus of TEMP to remove the putative [DE]XXX[LI] endosome sorting motif did not alter the distribution of the TEMP between endosomes and the plasma membrane
Summary
Determination of the subcellular distribution of individual proteins is essential to understanding a proteins function. We have performed several high-throughput subcellular localization projects on subsets of mouse proteins defined as part of the FANTOM project to determine the transcriptional output from the mouse genome [1±3]. For many of these proteins it represented the first property of these proteins to be determined and many displayed subcellular distributions of interest. We report the initial characterisation of a novel protein TEMP, a Type III Endosome Membrane Protein that displayed a plasma membrane and intracellular punctate subcellular localisation phenotype in the high throughput subcellular localisation screen [1]. Type III membrane proteins have a single membrane-spanning domain that acts as a reverse signal-anchor which results in the translocation of the amino-terminus across the membrane [7]
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