Study of the Plasma Membrane Proteome Dynamics Reveals Novel Targets of the Nitrogen Regulation in Yeast

Abstract

Yeast cells, to be able to grow on a wide variety of nitrogen sources, regulate the set of nitrogen transporters present at their plasma membrane. Such regulation relies on both transcriptional and post-translational events. Although microarray studies have identified most nitrogen-sensitive genes, nitrogen-induced post-translational regulation has only been studied for very few proteins among which the general amino acid permease Gap1. Adding a preferred nitrogen source to proline-grown cells triggers Gap1 endocytosis and vacuolar degradation in an Rsp5-Bul1/2-dependent manner. Here, we used a proteomic approach to follow the dynamics of the plasma membrane proteome after addition of a preferred nitrogen source. We identified new targets of the nitrogen regulation and four transporters of poor nitrogen sources-Put4, Opt2, Dal5, and Ptr2-that rapidly decrease in abundance. Although the kinetics is different for each transporter, we found that three of them-Put4, Dal5, and Ptr2-are endocytosed, like Gap1, in an Rsp5-dependent manner and degraded in the vacuole. Finally, we showed that Gap1 stabilization at the plasma membrane, through deletion of Bul proteins, regulates the abundance of Put4, Dal5 and Ptr2.

Conformation-dependent partitioning of yeast nutrient transporters into starvation-protective membrane domains

“…Several reports are also consistent with our finding that the number of EMCs and the area they occupy increase during nutrient limitation. For instance, the core eisosomal proteins (Pil1, Lsp1, Nce102, and Sur7) display a threefold increase in PM extracts from cells shifted to a poor nitrogen source (47). It has also long been shown by electron microscopy, in both budding and fission yeast, that in the stationary phase the PM invaginations increase in both number and depth (48,49), as is consistent with our observed increase in Pil1-mCherry intensity in the EMCs.…”

Fungal plasma membrane domains

“…Consistently, MCCs themselves are regulated by nutrient availability. Their number at the plasma membrane is low in nitrogen rich conditions and increases in nitrogen poor conditions or nitrogen starvation , consistent with a 3-fold difference in protein levels (Villers et al 2017). The more pronounced increase in MCCs, however is seen as cells reach the SP.…”

The yeast arrestin-related protein Bul1 is a novel actor of glucose-induced endocytosis

“…Strikingly, the deletion of BUL1 in the rod1∆ background almost completely abolished Jen1-GFP internalization ( Figure 1A ). Bul1 and Bul2 share many functions ( Helliwell et al , 2001 ; Soetens et al , 2001 ; Abe and Iida, 2003 ; Pizzirusso and Chang, 2004 ; Liu et al , 2007 ; Nikko and Pelham, 2009 ; Merhi and André, 2012 ; Crapeau et al , 2014 ; Villers et al , 2017 ), yet BUL2 deletion had no effect on Jen1 sorting ( Figure 1, A and B ), indicating that Bul1 alone is responsible for the “remnant internalization” reported earlier in the rod1∆ mutant ( Becuwe and Leon, 2014 ). Jen1-GFP trafficking was not altered in the single bul1 ∆ mutant, confirming that Rod1 remains the major ART for the glucose-induced endocytosis of Jen1 ( Figure 1C ).…”