Enhancement of Procollagen Biosynthesis by p180 through Augmented Ribosome Association on the Endoplasmic Reticulum in Response to Stimulated Secretion

Tomonori Ueno,Keisuke Tanaka,Keiko Kaneko,Yuki Taga,Tetsutaro Sata,Shinkichi Irie,Shunji Hattori,Kiyoko Ogawa-Goto

doi:10.1074/jbc.m109.094607

Abstract

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.

Highlights

We found that p180 depletion causes reduced activity of protein biosynthesis on the endoplasmic reticulum (ER), without significant reduction of the membranes or mRNAs
Elevation of the p180 levels by ascorbate stimulation or overexpression resulted in a concomitant increase in membrane-bound ribosomes
These data suggest that p180 is directly relevant to the ribosome association with the ER membranes

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Short Interfering RNA (siRNA) Oligonucleotide Transfection—HEL fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui) supplemented with 10% fetal bovine serum (FBS) (Intergen). The medium was replaced with DMEM/F12 containing 2% FBS and subjected to the following analyses at 3–5 days post-transfection. Electron Microscopy (EM)—For transmission EM analysis, cells cultured in 0.1% FBS/DMEM were treated with medium containing 10 ␮M taxol for 3 min at 37 °C, followed by a conventional fixation procedure for transmission EM as described previously [22]. Detergent extractions of HeLa cells were performed using permeabilization buffer containing 0.05% digitonin for 10 min on ice, followed by the same procedure as described above. To estimate newly synthesized proteins secreted into culture medium by pulse label experiments, cells preincubated with L-methionine-free medium for 1 h were cultured for 4 h in the presence of AHA (final concentration, 50 ␮M). Cells were extensively washed with PBS, and the culture medium was replaced with fresh DMEM containing 0.1% FBS. Specificity of metabolic labeling of newly synthesized proteins with AHA was assessed by comparing biotintagged protein patterns in the presence and absence of cycloheximide, or those in cultured with methionine instead of AHA (supplemental Fig. S3)

RESULTS

Findings

DISCUSSION