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(Received for publication, June 11, 1996, and in revised form, August 19, 1996)
From the Departments of Cell and Molecular Biology, The Scripps
Research Institute, La Jolla, California 92037
The small GTPase Rab2 is a resident of pre-Golgi
intermediates and required for protein transport from
the endoplasmic reticulum (ER) to the Golgi complex (Tisdale, E. J., Bourne, J. R., Khosravi-Far, R., Der, C. J., and Balch, W. E. (1992) J. Cell Biol. 119, 749-761). The Rab2 protein, like
all small GTPases, contains conserved GTP-binding domains as well as
hypervariable carboxyl-terminal and amino-terminal domains. While the
role of the carboxyl terminus in specific membrane localization is well
recognized, the potential role of the variable NH2 terminus
remains to be clarified. To determine whether the NH2
terminus of Rab2 was required for its activity in vivo, a trans dominant mutant of Rab2 that inhibits ER to Golgi transport was
progressively truncated and analyzed for its effect on vesicular stomatitis virus glycoprotein transport in a vaccinia-based transient expression system. Deletion of the first 14 amino-terminal residues resulted in the loss of the inhibitory properties of the mutant without
affecting its post-translational processing or membrane association. To
assess the potential role of the NH2 terminus in Rab2
function, a peptide corresponding to the first 13 amino acids following
the initiator methionine was introduced into an in vitro
assay that efficiently reconstitutes transport of vesicular stomatitis
virus glycoprotein from the ER to the Golgi stack. This peptide was a
potent inhibitor of transport. Biochemical and morphological studies
revealed that the peptide strongly interfered with assembly of
pre-Golgi intermediates which mediate segregation of anterograde and
retrograde transported proteins en route to the Golgi. The
combined results suggest that the NH2 terminus of Rab2 is
required for its function and for direct interaction with components of
the transport machinery involved in the maturation of pre-Golgi
intermediates.
Volume 271, Number 46,
Issue of November 15, 1996
pp. 29372-29379
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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