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Identification and characterization of GRIP domain Golgin PpImh1 from Pichia pastoris

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Budding yeast Pichia pastoris has highly advanced secretory pathways resembling mammalian systems, an advantage that makes it a suitable model system to study vesicular trafficking. Golgins are large Golgi‐resident proteins, primarily reported to play role in cargo vesicle capture, but details of such mechanisms are yet to be deciphered. Golgins that localize to the Golgi via their GRIP domain, a C‐terminal Golgi anchoring domain, are known as GRIP domain Golgins. In this present study, we have identified and functionally characterized a homologue of one such GRIP domain Golgin protein, Imh1, from the budding yeast P. pastoris. We have demonstrated that the GRIP domain present at the C‐terminal of P. pastoris Imh1 (PpImh1) functions as its Golgi‐targeting sequence. Using a combination of yeast two‐hybrid analysis, dynamic light scattering and electron microscopy, we have shown that PpImh1 can self‐associate and form a homodimer. Analysis of purified recombinant PpImh1 by CD spectroscopy indicates the presence of an 85% α‐helical structure, a characteristic of high‐content α‐helical coiled‐coil sequences normally present in other Golgin family proteins. Two‐hybrid analysis indicated self‐interaction between C‐terminal fragments, yet N‐terminal fragments do not mediate any such form of self‐interaction, suggesting that PpImh1 may form a parallel dimer. Electron microscopy data indicates that PpImh1 forms extended rod‐like homo‐dimeric molecules with splayed N‐terminal end which can act as a tether for capturing vesicles. Our study provides the first evidence in support of the dimeric Y‐shaped structure for any Golgin in the budding yeast.
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Keywords: GRIP domain; Golgi; Pichia pastoris; PpImh1

Document Type: Research Article

Publication date: August 1, 2018

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