Impediments to secretion of green fluorescent protein and its fusion from Saccharomyces cerevisiae.
While it has been demonstrated that GFP-tagged proteins were transported to their correct cellular compartments in most cells, attempts to secrete GFP/GFP-fusion through the default secretory pathway have not been as successful. In an attempt to induce secretion of GFP and Hexokinase (HXK)-GFP fusion in Saccharomycescerevisiae, we have cloned constructs that employed four different yeast secretion signal sequences, i.e., INU1, SUC2, PHO5, and MEL1. The expression is under the control of the galactose-inducible GAL1 promoter. Our results showed that all eight constructs entered the secretory pathway successfully, and the signal peptides were all cleaved off. However, none of the eight constructs were able to lead to secretion into the culture media or the periplasmic space. The expression levels of the eight constructs differ dramatically, depending on both the signal peptide and whether GFP was fused with HXK. Confocal microscopy studies revealed that the eight constructs also led to very different localization patterns. Among them, two constructs targeted GFP to the vacuole partially or exclusively, whereas others were mostly retained in the ER/Golgi compartments. Our efforts, together with those of others, seem to suggest that the signal peptide itself is not enough to lead to secretion of GFP from S. cerevisiae, although it has been successful in some other organisms. Nonetheless, the advantage of GFP's in vivo detection makes it a powerful tool for investigating protein localization events.
Li
Jincai
J
Department of Chemical and Biochemical Engineering and Microscopy Facility, Molecular and Cellular Biology Program, University of Maryland, Baltimore County, Maryland 21250, USA.
Xu
Haixin
H
Bentley
William E
WE
Rao
Govind
G
D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D004274:DNA, Recombinant / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005696:Galactosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006593:Hexokinase / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D043324:beta-Fructofuranosidase
D015415:Biomarkers; D004274:DNA, Recombinant; D005656:Fungal Proteins; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006593:Hexokinase; D000135:Acid Phosphatase; D005696:Galactosidases; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; C023086:inulinase