In situ cleavage of the acidic domain from the p115 tether inhibits exocytic transport

Abstract

Golgins are coiled-coil proteins involved in Golgi architecture and function. A complex of golgins (p115, GM130 and giantin), together with the rab1 guanosine triphosphatase and cis Golgi SNAREs, helps to mediate fusion processes at the entry face of the Golgi apparatus. The C-terminal acidic domain of p115 binds specifically to GM130 and giantin. However, deletion of this domain in vivo appears to have no effect on exocytic transport when using an RNA interference depletion/rescue approach (Puthenveedu MA, Linstedt AD. Gene replacement reveals that p115/SNARE interactions are essential for Golgi biogenesis. Proc Natl Acad Sci U S A 2004;101:1253-1256). In this study, we have used a different approach introducing a tobacco etch virus (tev) protease cleavage site into p115 so that the C-terminal domain can be rapidly and specifically released in vivo by microinjection of the tev protease. The results show that cleavage inhibits exocytic transport to the cell surface.

Figure 1. Schematic of p115 modified with the tev protease recognition site

Wild-type p115 comprises an N-terminal globular head domain (H), followed by a putative coiled-coil tail (T), ending in a C-terminal (CT) acidic domain. Binding partners and the mapped binding sites on p115 are indicated. The tev protease recognition site (ENLYFQG) was inserted between the tail and CT after position 886. For detection purposes, the constructs had either an N-terminal HA- and/or a C-terminal Myc-tag.

Figure 2. Cleavage of modified p115 in vitro

Wild-type p115 (p115) and tev-modified p115 (p115(tev)), tagged at both ends (HA at the N-terminus, Myc at the C-terminus), were stably expressed in HeLa cells. An N-terminal fragment (HT, 1–886; HA-tagged at the N-terminus) was also stably expressed. Tagged proteins were immuno-precipitated from cell lysates using anti-HA-agarose, treated with (+) or without (−) tev protease, and fractionated by SDS-PAGE followed by Western blotting (WB) with anti-HA (A; detection of N-termini), with anti-Myc (B; detection of C-termini) or with anti-CT antibodies (C and D (longer exposure); detection of cleaved CT fragment (arrow in D) and endogenous p115 (asterisk in D)). E. Fractionated samples were also probed by Far Western blotting using the N-terminal 73 amino acids of GM130 fused to GST (GM130N73-GST). Arrowheads in panels B, C and D indicate protein standards (kDa, from top): 150, 100, 75, 50, 37.5, 25, 20 and 10kDa. # indicates the position of HT.

Figure 3. Cleavage of modified p115 in vivo

HeLa cells stably expressing p115 or p115(tev), tagged at both ends (HA at N-terminus, Myc at the C-terminus), were micro-injected with tev protease (asterisks in right panels). After 2 h at 37°C, cells were fixed, permeabilized and labeled with (left panels) anti-Myc antibodies and either (top, middle panels) anti-p115T (which recognizes the coiled-coil tail, not the CT domain of p115), or (bottom, middle panels) the Golgi marker, giantin. The merged images are on the right (Myc: green, other markers: red). Cell boundaries outlined in white. Bar, 20 µm.

Figure 4. Cleavage of modified p115 in vivo inhibits cargo transport

(A) HeLa cells transiently expressing p115 or p115(tev) were micro-injected (asterisks), in the presence of cycloheximide, with the cDNA encoding a cell-surface marker, CD8, together with the tev protease. After 1 h at 37 °C to accumulate CD8 mRNA, the cycloheximide was removed, and cells incubated for 2.5 h at 37 °C with a 45-min cycloheximide chase at the end of the incubation. Cells were fixed, permeabilized and labeled with anti-CD8 polyclonal antibodies to visualize total CD8. Arrows mark the cell periphery. Bar, 20 µm. (B) HeLa cells stably expressing p115 or p115(tev), or transfected with the vector alone (ctrl), were micro-injected as in (A). Cells were fixed and labeled for surface and total CD8, the ratio being determined for each cell. The results were normalized to the mean value of p115(tev) without protease, set to 100%, and expressed as the mean ± SD (n=15~20 cells). C. HeLa cells were micro-injected with cDNA encoding CD8 and either GST or GST-CT (~3 mg/ml) and processed as in B. The results were normalized to the mean value of GST, set to 100%, and expressed as the mean± SD (n=15~20 cells).