Abstract
Although all mammalian COPII components have now been cloned, little is known of their interactions with other regulatory proteins involved in exit from the endoplasmic reticulum (ER). We report here that a mammalian protein (Yip1A) that is about 31% identical to S. cerevisiae and which interacts with and modulates COPII-mediated ER-Golgi transport. Yip1A transcripts are ubiquitously expressed. Transcripts of a related mammalian homologue, Yip1B, are found specifically in the heart. Indirect immunofluorescence microscopy revealed that Yip1A is localized to vesicular structures that are concentrated at the perinuclear region. The structures marked by Yip1A co-localized with Sec31A and Sec13, components of the COPII coat protein complex. Immunoelectron microscopy also showed that Yip1A co-localizes with Sec13 at ER exit sites. Overexpression of the hydrophilic N terminus of Yip1A arrests ER-Golgi transport of the vesicular stomatitis G protein and causes fragmentation and dispersion of the Golgi apparatus. A glutathione S-transferase fusion protein with the hydrophilic N terminus of Yip1A (GST-Yip1A) is able to bind to and deplete vital components from rat liver cytosol that is essential for in vitro vesicular stomatitis G transport. Peptide sequence analysis of cytosolic proteins that are specifically bound to GST-Yip1A revealed, among other proteins, mammalian COPII components Sec23 and Sec24. A highly conserved domain at the N terminus of Yip1A is required for Sec23/Sec24 interaction. Our results suggest that Yip1A is involved in the regulation of ER-Golgi traffic at the level of ER exit sites.
Highlights
IntroductionMolecular and biochemical characterization of the cellular components (for recent reviews, see Refs. 1 and 2) responsible for the formation of transport vesicles from the ER1 is impor-
Molecular and biochemical characterization of the cellular components responsible for the formation of transport vesicles from the ER1 is impor
The ER exits sites (ERES) are located in discrete positions of the endoplasmic reticulum (ER) reticulum and are found with the highest density at the perinuclear region, presumably where the ER membranes are in close apposition to the Golgi apparatus
Summary
Molecular and biochemical characterization of the cellular components (for recent reviews, see Refs. 1 and 2) responsible for the formation of transport vesicles from the ER1 is impor-. Antibodies against the mammalian COPII components (17, 19 –21, 25) provided the first morphological definition of COPII-budding sites, or ER exits sites (ERES) [29], on specific regions of the ER. These appear as vesicular “spotty” structures by light microscopy. Immunogold labeling of COPII components can be found in distinct vesiculotubular structures devoid of ribosomes These structures appear to be similar to structures where several viral proteins accumulate upon a low temperature (15 °C) treatment and which had been defined in terms such as vesicle-tubular structures [30] and the ER-Golgi intermediate compartment [31]
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