Role of VAMP-2, VAMP-7, and VAMP-8 in constitutive exocytosis from HSY cells.
We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent signal of hGH-GFP was observed in the Golgi area and small vesicles quickly moving throughout the cytoplasm. The cytoplasmic vesicles containing hGH overlapped well with VAMP-7-GFP, but did so scarcely with VAMP-2-GFP or VAMP-8-GFP. However, when the vesicle transport from the trans-Golgi network to the plasma membrane was arrested by incubation at 20 degrees C for 2 h and then released by warming up to 37 degrees C; VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH-GFP was colocalized with LAMP-1, a marker for lysosomes and late endosomes. These results suggest that (1) VAMP-2 can be one of the v-SNAREs for constitutive exocytosis; (2) VAMP-7 is involved in the constitutive exocytosis as a slow, minor v-SNARE, but not in the lysosomal transport; and (3) VAMP-8 is unlikely to be a v-SNARE for constitutive exocytosis in HSY cells.
Oishi
Yohei
Y
Department of Oral Biochemistry, School of Dentistry, Health Sciences University of Hokkaido, Tobetsu, Hokkaido, 061-0293 Japan.
Arakawa
Toshiya
T
Tanimura
Akihiko
A
Itakura
Makoto
M
Takahashi
Masami
M
Tajima
Yoshifumi
Y
Mizoguchi
Itaru
I
Takuma
Taishin
T
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004847:Epithelial Cells / Q000502:physiology; D005089:Exocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D019382:Human Growth Hormone / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D010306:Parotid Gland / Q000502:physiology; D050683:R-SNARE Proteins / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000502:physiology*
D017931:DNA Primers; D050683:R-SNARE Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C494807:VAMP2 protein, human; C121241:VAMP7 protein, human; C112938:VAMP8 protein, human; D050988:Vesicle-Associated Membrane Protein 2; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins