Golgi matrix proteins interact with p24 cargo receptors and aid their efficient retention in the Golgi apparatus

Abstract

The Golgi apparatus is a highly complex organelle comprised of a stack of cisternal membranes on the secretory pathway from the ER to the cell surface. This structure is maintained by an exoskeleton or Golgi matrix constructed from a family of coiled-coil proteins, the golgins, and other peripheral membrane components such as GRASP55 and GRASP65. Here we find that TMP21, p24a, and gp25L, members of the p24 cargo receptor family, are present in complexes with GRASP55 and GRASP65 in vivo. GRASPs interact directly with the cytoplasmic domains of specific p24 cargo receptors depending on their oligomeric state, and mutation of the GRASP binding site in the cytoplasmic tail of one of these, p24a, results in it being transported to the cell surface. These results suggest that one function of the Golgi matrix is to aid efficient retention or sequestration of p24 cargo receptors and other membrane proteins in the Golgi apparatus.

Figure 1.

GRASPs and p24 proteins form a complex in Golgi membranes. (A) GRASP55 complexes were isolated from detergent extracts of Golgi membranes. A 10% Coomassie blue–stained minigel run under nonreducing conditions is shown; loadings corresponding to 1/30 and 29/30 of the total isolated material. Bands corresponding to the specifically interacting proteins seen in all experiments performed (n = 5) were excized (arrowheads and bracket), and tryptic digests of the proteins contained therein analyzed by mass spectrometry. Asterisks indicate rat serum albumin confirmed by mass spectrometry and sequencing of tryptic peptides. The total eluted material from a minus antibody control is also shown. (B) Immunoprecipitations (IPs) were performed from 200 μg Golgi membranes with the following antibodies: sheep anti-GRASP55, mouse anti-GRASP65, rabbit anti-golgin-45, and sheep anti-GM130. All IPs were blotted with rabbit antibodies to p24a and gp25L. (C) Blots with the following antibodies were performed as controls: GRASP55 and GRASP65 IPs with rabbit anti-GM130, and rabbit anti–golgin-45; GM130 IPs with sheep anti-GRASP55 and mouse anti-GRASP65; golgin-45 IPs with sheep anti-GRASP55 and mouse anti-GRASP65. The asterisk indicates cross-reactivity to the heavy chain of the sheep anti-GM130 antibody.

Figure 2.

A direct interaction between some p24 cytoplasmic tails and GRASPs. (A) GRASP55 and (B) GRASP65 were screened against the cytoplasmic tails of the p24 proteins indicated, TGF-α, and GM130 either with or without a coiled-coil sequence to mediate oligomerization. Interactions were selected by the ability to grow on media lacking leucine, tryptophan, histidine, and adenine (QDO) after initial growth on minimal media lacking leucine, tryptophan and adenine (-LWA). (C) GRASP55 and GRASP65 two-hybrid interactions were measured using the lac-Z reporter gene. GM130 (-cc) is the monomeric form of the signal lacking the coiled coil. (D) Binding of purified GRASP55 and GRASP65 to GST fusion proteins bearing the cytoplasmic tails of the various p24 proteins, TGF-α, and GM130. Background binding of GRASPs to the beads is also shown (control).

Figure 3.

A GRASP binding motif exists in the p24a cytoplasmic tail. (A) GRASP55 and (B) GRASP65 were screened against sequences corresponding to the cytoplasmic tail of wild-type and point mutant forms of p24a either without (monomer) or with (oligomer) a coiled-coil sequence to mediate oligomerization. Interactions were tested for by selection on QDO after initial growth on minimal media lacking leucine and tryptophan (-LW). (C) GRASP55 and GRASP65 two-hybrid interactions were quantitated using the lac-Z reporter gene. (D) Binding of purified GRASP55 and GRASP65 to GST fusion proteins bearing the wild-type and point mutant forms of the p24a cytoplasmic tail, and GM130. Background binding of GRASPs to the beads is also shown (control).

Figure 4.

The GRASP binding motif is needed for efficient Golgi retention of p24a. (A) Cells were transfected for 18 h with plasmids encoding GFP-tagged p24a with either the wild-type cytoplasmic tail, or mutants abolishing the GRASP binding site, VV→AA, the phenylalanine and arginine motifs, FF→AA and RR→AA, or combinations thereof, FFVV→AA and RRVV→AA. Total fluorescence was visualized with GFP, and cell-surface fluorescence by an antibody to GFP detected with a CY3-coupled secondary antibody. (B) Extent of p24a transport to the cell surface was measured for wild-type and mutant p24a tails constructs. This ratio does not approach unity due to the different dyes used to measure surface and total fluorescence. Mean and standard deviation are plotted, n = 20. Bar, 20 μm