The movement of newly synthesized membrane protein through the
complex array of intracellular membranes presents interesting questions
of both sorting and motility. Recently, it has been possible to
tag newly synthesized protein naturally with a fluorescent
tag by inserting the nucleotide sequence for Green Fluorescent Protein
(GFP, a jelly fish protein) to one end of the gene coding for the
protein of interest. Following translation, GFP spontaneously folds
into a fluorescent tag that in many instances does not
inhibit the subsequent processing or ultimate structure or function
of its chimeric partner.
The figures and videos that follow were produced by Presley, et
al.(1998. Mol. Biol. Cell. 9:1617-1626. PMID9658158) from time-lapse
observations of the progression of newly synthesized, GFP-labeled
vesicular stomatitis virus membrane protein(GFP-VSVP) from ER to
the Golgi complex in cultured COS cells. Very thin optical sections
of each cell were obtained by confocal fluorescence microscopy.
In these images, a faintly fluorescing ER network surrounds a collection
of much brighter, large vesicles that constitutes the Golgi complex.
In all instances the Golgi complex, when present, is situated adjacent
to the nucleus. Images in the first video were captured by a digital
camera about every 9 sec; they are displayed in the quick-time movie
at a rate of about 9 frames a second and thus are speeded up about
81-fold. Recall what you know about the steps of post-translational
processing, click on the still image below to bring up the movie,
watch the movement of GFP-VSVP into the Golgi several times (at
least once, one frame at a time) and answer the questions in the
right-hand frame.
[You can learn more about these phenomena and the following
experiments by consulting the video essay by Presley, et al. 1998
Mol Biol. Cell 9:1617-1626. PMID9658158]
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Questions
A. Describe carefully the movement of GFP-VSVP with respect to the
Golgi complex.
B. Critically compare your observations here with your expectations
based on text and lecture.
C. To obtain more information about this process, cells were cooled
for several hours, which retarded translocation more than translation.Consequently,
newly translated GFP-VSVP accumulated in pre-Golgi regions prior
to heating. Turn the page to view the results
of this experiment.
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