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2016 ; 4
(ä): 13
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Identification of Rab41/6d Effectors Provides an Explanation for the Differential
Effects of Rab41/6d and Rab6a/a on Golgi Organization
#MMPMID26973836
Liu S
; Majeed W
; Kudlyk T
; Lupashin V
; Storrie B
Front Cell Dev Biol
2016[]; 4
(ä): 13
PMID26973836
show ga
Unexpectedly, members of the Rab VI subfamily exhibit considerable variation in
their effects on Golgi organization and trafficking. By fluorescence microscopy,
neither depletion nor overexpression of the GDP-locked form of Rab6a/a', the
first trans Golgi-associated Rab protein discovered, affects Golgi ribbon
organization while, on the other hand, both Rab41/6d depletion and overexpression
of GDP-locked form cause Golgi fragmentation into a cluster of punctate elements,
suggesting that Rab41/6d has an active role in maintenance of Golgi ribbon
organization. To establish a molecular basis for these differences, we screened
for Rab41/6d interacting proteins by yeast two-hybrid assay. 155 non-repetitive
hits were isolated and sequenced, and after searching in NCBI database, 102
different proteins and protein fragments were identified. None of these hits
overlapped with any published Rab6a/a' effector. Eight putative Rab41 interactors
involved in membrane trafficking were found. Significantly, these exhibited a
preferential interaction with GTP- vs. GDP-locked Rab41/6d. Of the 8 hits, the
dynactin 6, syntaxin 8, and Kif18A plasmids were the only ones expressing the
full-length protein. Hence, these 3 proteins were selected for further study. We
found that depletion of dynactin 6 or syntaxin 8, but not Kif18A, resulted in a
fragmented Golgi apparatus that displayed a Rab41/6d knockdown phenotype, i.e.,
the Golgi apparatus was disrupted into a cluster of punctate Golgi elements.
Co-immunoprecipation experiments verified that the interaction of dynactin 6 and
syntaxin 8 with GTP-locked Rab41/6d was stronger than that with wild type
Rab41/6d and least with the GDP-locked form. In contrast, co-immunoprecipitation
interaction with Rab6a was greatest with the GDP-locked Rab6a, suggestive of a
non-physiological interaction. In conclusion, we suggest that dynactin 6, a
subunit of dynactin complex, the minus-end-directed, dynein motor, provides a
sufficient molecular basis to explain the active role of Rab41/6d in maintaining
Golgi ribbon organization while syntaxin 8 contributes more indirectly to Golgi
positioning.