Reduction in Golgi apparatus dimension in the absence of a residential protein, N-acetylglucosaminyltransferase V

Abstract

Various proteins are involved in the generation and maintenance of the membrane complex known as the Golgi apparatus. We have used mutant Chinese hamster ovary (CHO) cell lines Lec4 and Lec4A lacking N-acetylglucosaminyltransferase V (GlcNAcT-V, MGAT5) activity and protein in the Golgi apparatus to study the effects of the absence of a single glycosyltransferase on the Golgi apparatus dimension. Quantification of immunofluorescence in serial confocal sections for Golgi α-mannosidase II and electron microscopic morphometry revealed a reduction in Golgi volume density up to 49 % in CHO Lec4 and CHO Lec4A cells compared to parental CHO cells. This reduction in Golgi volume density could be reversed by stable transfection of Lec4 cells with a cDNA encoding Mgat5. Inhibition of the synthesis of β1,6-branched N-glycans by swainsonine had no effect on Golgi volume density. In addition, no effect on Golgi volume density was observed in CHO Lec1 cells that contain enzymatically active GlcNAcT-V, but cannot synthesize β1,6-branched glycans due to an inactive GlcNAcT-I in their Golgi apparatus. These results indicate that it may be the absence of the GlcNAcT-V protein that is the determining factor in reducing Golgi volume density. No dimensional differences existed in cross-sectioned cisternal stacks between Lec4 and control CHO cells, but significantly reduced Golgi stack hits were observed in cross-sectioned Lec4 cells. Therefore, the Golgi apparatus dimensional change in Lec4 and Lec4A cells may be due to a compaction of the organelle.

Fig. 1

Demonstration of β1,6-branched N-glycans on the CHO cell surface using dig L-PHA and confocal laser scanning microscopy (a–c) and lectin-gold scanning electron microscopy (e–g). The CHO Pro⁻5 cells (a, e) and CHO Lec4 GnTV-N5 cells (c, g) showed positive cell surface staining with microvilli being strongly positive. In contrast, CHO Lec4 cells (b, f) showed no specific cell surface labeling. ×1,100 (a–c), ×9,000 (e–g). Dig L-PHA—blot of homogenates from CHO Lec4 GnTV cells revealed two major bands at 140 and 85 kDa and several minor bands (d)

Fig. 2

Confocal laser scanning immunofluorescence for Golgi α-mannosidase II. CHO Pro⁻5 cells (a), CHO Lec4 cells (b), and CHO Lec4 GnTV-N5 cells (c) exhibit a typical perinuclear Golgi staining pattern in a single confocal optical section (×560)

Fig. 3

Transmission electron micrographs showing the morphology of the Golgi apparatus (GA) in CHO Pro⁻5 cells (a), CHO Lec4 cells (b), and CHO Lec4 GnTV-N5 cells (c). Note the structural similarity in appearance of the Golgi apparatus in the different cells lines. N part of the nucleus (×41,300)

Fig. 4

The distribution pattern of volume density of the Golgi apparatus in the various CHO cell lines. The number of cross-sectioned cells containing no cisternal stacks was much higher in CHO Lec4 cells than in CHO Pro⁻5 as well as CHO Lec4 GnTV-N5 cells

Fig. 5

Reduction in the β1,6-branched N-glycans in CHO cells after swainsonine treatment. Swainsonine (1 μg/ml) was added to the cell culture medium, and cells were collected after 0, 12, and 24 h of treatment. Homogenates were processed for L-PHA spot blots, and the results densitometrically analyzed. The CHO Lec4 cells served as control