Bmh1-specific function in protein transport along the secretory pathway
To better understand the specific role of Bmh1 in membrane transport of the YFP reporter we assessed this phenomenon quantitatively by determining the subcellular localisation of the reporter in wild type, bmh1 and bmh2 knockout strains (Fig. 1A, B). In logarithmic phase, the reporter was predominately localised to the vacuole of wild type and bmh2 cells (left panels (A)) in ca. 80% of cells, quantification in (B). In contrast, the reporter localised to the vacuole of only 30% and was visible in the ER of 65% of bmh1 cells, suggesting that trafficking of our YFP reporter along the secretory pathways was compromised. These phenotypes became even more pronounced during stationary phase. Since stationary yeast cultures typically experience glucose deprivation we assayed whether this was the relevant parameter determining the Bmh1 requirement for reporter localisation. Indeed, upon glucose starvation, the requirement for Bmh1 was even more pronounced and the reporter only fully reached the vacuole in 10% of cells, as opposed to 50% in the wild type and bmh2 deletion strains (Fig. 1A, B). We concluded that the role of the 14-3-3 Bmh1 in targeting the YFP reporter to the vacuole is particularly relevant under conditions of glucose starvation.
Bmh1 but not Bmh2 is required to maintain the global morphology of the endomembrane system
To assess the global morphology of the endomembrane system, we investigated the wild type, bmh1 and bmh2 deletion strains after glucose starvation using transmission electron microscopy (TEM) (Fig. 1C, E, G). Qualitative assessment revealed three different subcellular structures whose abundance varied with the genotypes, that is, multivesicular bodies (MVBs, Fig. 1C), vesicle clusters (VC, Fig. 1E) and glycogen granules (Fig. 1G). Quantification revealed that the bmh1 strain possessed less MVBs (Fig. 1D) and less VCs (Fig. 1F), consistent with global alterations in vesicular traffic. Furthermore, we observed substantial accumulation of glycogen granules in the bmh1 strain (Fig. 1H), which was independently confirmed by iodine staining (Fig. 1I).
The guanine nucleotide exchange factor Gea1 rescues Bmh1-specific alterations in the endomembrane system/vesicular transport but not glycogen accumulation.
Guanine nucleotide exchange factor (GEF) proteins activate the GTPase Arf1 by conversion of GDP-Arf1 to active GTP-Arf1. In consequence, the N-terminal myristoylation moiety is exposed and is able to anchor Arf1 in the Golgi membrane, where it recruits COPI coat components and initiates the process of COPI budding. Gea1 is one of four GEFs found in yeast. Together with mammalian GBF1, Gea1 and Gea2 are part of a subfamily of GEFs distinct from the Sec7 subfamily. Different functionalities of the subfamilies are highlighted by the fact that GEA1 cannot suppress the temperature sensitivity of a sec7 mutant and vice versa. Phosphorylation of GBF1 was shown to have a critical role in Golgi disassembly and gea1/2 mutants showed disrupted Golgi in yeast, supporting the notion that they have similar roles in the two systems. Furthermore, Gea1 has been shown to increase the rate of GTPɣS binding to Arf1 in vitro suggesting that it may affect COPI-dependent processes in vivo.
In mammalian cells, AMPK kinase is known to phosphorylate the GEF GBF1. It is thought that this regulation attenuates the function of GBF1 when the intracellular concentration of ATP drops. Therefore, yeast AMPK may regulate Gea1 function in response to glucose availability. Hence, we tested whether we could suppress Bmh1-specific phenotypes by overexpression of GEA1, to date the better studied of the two yeast homologues of GBF1, to supply the cell with additional GEF activity and thus counteract a putative down-regulation of GEF function in the bmh1 deletion strain (Fig. 1J, K, L, M). Indeed, overexpression of GEA1 restored the localisation of the YFP reporter to the vacuole in the bmh1 strain (Fig. 1M), just as observed for the wild type and bmh2 strains. Compare figure 1A right, without GEA1 overexpression and figure 1M showing a cell in which GEA1 was overexpressed. Similarly, GEA1 overexpression in the bmh1 strain increased the abundance of MVBs (Fig. 1J) and VCs (Fig. 1K) to levels even higher than in the wild type strain. In contrast, the accumulation of glycogen granules in the bmh1 background was unaffected by overexpression of the Arf-GEF GEA1 (Fig. 1L). These results are consistent with the notion that the global alterations in vesicular traffic observed in the bmh1 strain (reduction of reporter trafficking to the vacuole, reduction in MVBs and VCs) are downstream of Gea1 function and that Gea1 is less functional in the absence of this 14-3-3 protein. However, overexpression of GEA1 did not suppress glycogen accumulation in the bmh1 strain. This indicates that Bmh1 action is upstream of Gea1 in a pathway that culminates in changes in vesicular trafficking, yet Bmh1 function is independent of Gea1 in the pathway leading to glycogen accumulation.