Islet Cell Autoantigen of 69 kDa Is an Arfaptin-related Protein Associated with the Golgi Complex of Insulinoma INS-1 Cells

Folker Spitzenberger,Susan Pietropaolo,Paul Verkade,Bianca Habermann,Sandra Lacas-Gervais,Hassan Mziaut,Massimo Pietropaolo,Michele Solimena

doi:10.1074/jbc.m213222200

Abstract

Islet cell autoantigen of 69 kDa (ICA69) is a cytosolic protein of still unknown function. Involvement of ICA69 in neurosecretion has been suggested by the impairment of acetylcholine release at neuromuscular junctions upon mutation of its homologue gene ric-19 in C. elegans. In this study, we have further investigated the localization of ICA69 in neurons and insulinoma INS-1 cells. ICA69 was enriched in the perinuclear region, whereas it did not co-localize with markers of synaptic vesicles/synaptic-like microvesicles. Confocal microscopy and subcellular fractionation in INS-1 cells showed co-localization of ICA69 with markers of the Golgi complex and, to a minor extent, with immature insulin-containing secretory granules. The association of ICA69 with these organelles was confirmed by immunoelectron microscopy. Virtually no ICA69 immunogold labeling was observed on secretory granules near the plasma membrane, suggesting that ICA69 dissociates from secretory granule membranes during their maturation. In silico sequence and structural analyses revealed that the N-terminal region of ICA69 is similar to the region of arfaptins that interacts with ARF1, a small GTPase involved in vesicle budding at the Golgi complex and immature secretory granules. ICA69 is therefore a novel arfaptin-related protein that is likely to play a role in membrane trafficking at the Golgi complex and immature secretory granules in neurosecretory cells.

Highlights

Islet cell autoantigen of 69 kDa (ICA69) is a cytosolic protein of still unknown function
In silico sequence and structural analyses revealed that the N-terminal region of ICA69 is similar to the region of arfaptins that interacts with ARF1, a small GTPase involved in vesicle budding at the Golgi complex and immature secretory granules
To map the epitope recognized by monoclonal antibodies against human ICA69 (mICA69), the N-terminal and C-terminal halves of ICA69 were independently expressed as 35S-labeled polypeptides by in vitro transcription/translation and incubated either with mICA69 or control mouse IgG for immunoprecipitation followed by SDS-PAGE and autoradiography (Fig. 1A)

Summary

Introduction

Islet cell autoantigen of 69 kDa (ICA69) is a cytosolic protein of still unknown function. Confocal microscopy and subcellular fractionation in INS-1 cells showed co-localization of ICA69 with markers of the Golgi complex and, to a minor extent, with immature insulin-containing secretory granules.

Results

Conclusion