GRASP65 and GRASP55 Sequentially Promote the Transport of C-terminal Valine-bearing Cargos to and through the Golgi Complex

Luisa Iodice,Giovanni D'Angelo,Stefano Bonatti,Maria Antonietta De Matteis,Giuseppe Di Tullio,Pierfrancesco Marra,Gianluca Martire,Libera Prencipe,Galina Beznoussenko,Marco Savarese

doi:10.1074/jbc.m109.068403

Abstract

The Golgi matrix proteins GRASP65 and GRASP55 have recognized roles in maintaining the architecture of the Golgi complex, in mitotic progression and in unconventional protein secretion whereas, surprisingly, they have been shown to be dispensable for the transport of commonly used reporter cargo proteins along the secretory pathway. However, it is becoming increasingly clear that many trafficking machineries operate in a cargo-specific manner, thus we have investigated whether GRASPs may control the trafficking of selected classes of cargo. We have taken into consideration the C-terminal valine-bearing receptors CD8alpha and Frizzled4 that we show bind directly to the PSD95-DlgA-zo-1 (PDZ) domains of GRASP65 and GRASP55. We demonstrate that both GRASPs are needed sequentially for the efficient transport to and through the Golgi complex of these receptors, thus highlighting a novel role for the GRASPs in membrane trafficking. Our results open new perspectives for our understanding of the regulation of surface expression of a class of membrane proteins, and suggests the causal mechanisms of a dominant form of autosomal human familial exudative vitreoretinopathy that arises from the Frizzled4 mutation involving its C-terminal valine.

Highlights

GRASP65 and GRASP55 were identified in in vitro assays as factors that are required for the stacking of the Golgi cisternae [1, 2]
We show that a similar mechanism operates for the Frizzled4 receptor (Fz4), which is a membrane-multispanning protein involved in a number of signaling events at the plasma membrane, and is associated with the human familial exudative vitroretinopathy (FEVR), a hereditary ocular disorder (26 –28)
We have shown that GRASP65 and GRASP55 decode the information provided by the C-terminal valine motifs (C-TVM) of CD8␣ and Fz4 to sequentially promote their anterograde transport along the secretory pathway

Summary

Role of GRASPs in Protein Transport

We have investigated here the possibility that the GRASPs may selectively control the transport of neosynthesized C-valine cargos. To this end we have combined the two independent approaches of removing the C-TVM and interfering with the GRASP machinery. We provide biochemical and functional evidence that GRASP65 and GRASP55 bind directly to newly synthesized CD8␣ in a C-TVM-dependent fashion and show that the GRASPs control two sequential transport steps of CD8␣ from the ER into the Golgi complex. Our results demonstrate a novel role for GRASPs in the transport of selected cargo along the conventional secretory pathway, and provide a molecular pathogenetic explanation for the defect underlying a dominant form of human FEVR, which is induced by mistrafficking of a mutated Fz4

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION