Ypt/Rab GTPases are conserved molecular switches that regulate the multiple vesicular transport steps of all intracellular trafficking pathways. They are stimulated by guanine-nucleotide exchange factors (GEFs). In yeast, Ypt1 regulates transport from the endoplasmic reticulum (ER) to two alternative pathways: secretion and autophagy. Ypt1 is activated by TRAPP, a modular multi-subunit GEF. Whereas TRAPP I activates Ypt1 to mediate transport through the Golgi, TRAPP III, which contains all the subunits of TRAPP I plus Trs85, activates Ypt1-mediated transport to autophagosomes. The functional pair Ypt31/32 regulates traffic in and out of the trans-Golgi and is activated by TRAPP II, which consists of TRAPP I plus two specific subunits, Trs120 and Trs130. To study the interaction of Ypts with specific TRAPP subunits and interactions between the different subunits of TRAPP, including the cellular sites of these interactions, we have employed a number of approaches. One approach that we have recently optimized for the use in yeast is multicolor bimolecular fluorescence complementation (BiFC). BiFC, which employs split fluorescent tags, has emerged as a powerful approach for determining protein interaction in vivo. Because proteins work in complexes, the ability to determine more than one interaction at a time using multicolor BiFC is even more powerful. Defining the sites of protein interaction is possible by co-localization of the BiFC puncta with compartmental markers. Here, we describe a set of plasmids for multicolor BiFC optimized for use in yeast. We combined their use with a set of available yeast strains that express red fluorescence compartmental markers. We have recently used these constructs to determine Ypt1 and TRAPP interactions in two different processes: intracellular trafficking and autophagy.

• PMID: 25800836
• DOI full text
• PMC full text
• PubMed
• PubTator

Reference Type

Journal Article | Research Support, N.I.H., Extramural

Authors

Lipatova Z, Kim JJ, Segev N

Primary Lit For

Additional Lit For

Review For

YPT10 | YPT7 | YPT1 | YPT31 | YPT52 | SEC4 | YPT32 | YPT6 | ATG11 | TRS85 | VPS21 | ... Show allYPT53 Show fewer