Rab3D, a small GTP-binding protein implicated in regulated secretion, is associated with the transcytotic pathway in rat hepatocytes

Janet M. Larkin, Bonnie Woo, Vijayan Balan, David L. Marks, Barbara J. Oswald, Nicholas F La Russo, Mark A Mc Niven

Research output: Contribution to journalArticle

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Abstract

Rab3 isotypes are expressed in regulated secretory cells. Here, we report that rab3D is also expressed in rat hepatocytes, classic models for constitutive secretion. Using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for rat rab3D, we amplified a 151 base pair rab3D fragment from total RNA extracted from primary cultures of rat hepatocytes. Immunoblot analysis using polyclonal antibodies to peptides representing the N- and C-terminal hypervariable regions of murine rab3D recognized a protein of ~25 kd in hepatocyte lysates, hepatic subcellular fractions, and tissue extracts. The distribution of rab3D was primarily cytosolic; however, only membrane-associated rab3D significantly bound guanosine triphosphate (GTP) in overlay assays. Several lines of investigation indicate that rab3D is associated with the transcytotic pathway. First, rab3D was enriched in a crude vesicle carrier fraction (CVCF), which includes transcytotic carriers. Vesicular compartments immunoisolated from the CVCF on magnetic beads coated with anti-rab3D antibody were enriched in the transcytosed form of the polymeric IgA receptor (pIgA-R), but lacked not only the pIgA-R precursor form associated with the secretory pathway, but also a Golgi marker protein. Second, indirect immunofluorescence on frozen liver sections and in polarized cultured hepatocytes localized rab3D-positive sites at or near the apical plasma membrane and to the pericanalicular cytoplasm. Finally, cholestasis induced by bile duct ligation (BDL), a manipulation known to slow transcytosis, caused rab3D to accumulate in the pericanalicular cytoplasm of cholestatic hepatocytes. Our results indicate that rab3D plays a role in the regulation of apically directed transcytosis in rat hepatocytes.

Original languageEnglish (US)
Pages (from-to)348-356
Number of pages9
JournalHepatology
Volume32
Issue number2
StatePublished - 2000

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Transcytosis
Guanosine Triphosphate
Hepatocytes
Carrier Proteins
Cytoplasm
Tissue Extracts
Subcellular Fractions
Secretory Pathway
Liver
Cholestasis
Frozen Sections
Indirect Fluorescent Antibody Technique
Bile Ducts
Reverse Transcriptase Polymerase Chain Reaction
Base Pairing
Ligation
Anti-Idiotypic Antibodies
Proteins
Cell Membrane
RNA

ASJC Scopus subject areas

  • Hepatology

Cite this

Rab3D, a small GTP-binding protein implicated in regulated secretion, is associated with the transcytotic pathway in rat hepatocytes. / Larkin, Janet M.; Woo, Bonnie; Balan, Vijayan; Marks, David L.; Oswald, Barbara J.; La Russo, Nicholas F; Mc Niven, Mark A.

In: Hepatology, Vol. 32, No. 2, 2000, p. 348-356.

Research output: Contribution to journalArticle

Larkin, Janet M. ; Woo, Bonnie ; Balan, Vijayan ; Marks, David L. ; Oswald, Barbara J. ; La Russo, Nicholas F ; Mc Niven, Mark A. / Rab3D, a small GTP-binding protein implicated in regulated secretion, is associated with the transcytotic pathway in rat hepatocytes. In: Hepatology. 2000 ; Vol. 32, No. 2. pp. 348-356.
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