The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p.

Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling

Arf GTPase regulation through cascade mechanisms and positive feedback loops

AbstractArf GTPases, together with Rab GTPases, are key regulators of intracellular membrane traffic. Their specific membrane targeting and activation are tightly regulated in time and space by guanine nucleotide exchange factors (GEFs). GEFs are multidomain proteins, which are under tight regulation to ensure fully coordinated and accurate membrane traffic events. Recently, two Arf GEFs, Sec7 and Arno, have been shown to be part of Arf GEF cascades similar to the Rab GEF cascades. Both GEFs are autoinhibited in solution and require an active Arf molecule to be recruited to the membrane and to switch to an open conformation. As such, positive feedback loops, whereby the amount of Arf-GTP on a given organelle increases not linearly with time, can be established

Direct trafficking pathways from the Golgi apparatus to the plasma membrane.

In eukaryotic cells, protein sorting is a highly regulated mechanism important for many physiological events. After synthesis in the endoplasmic reticulum and trafficking to the Golgi apparatus, proteins sort to many different cellular destinations including the endolysosomal system and the extracellular space. Secreted proteins need to be delivered directly to the cell surface. Sorting of secreted proteins from the Golgi apparatus has been a topic of interest for over thirty years, yet there is still no clear understanding of the machinery that forms the post-Golgi carriers. Most evidence points to these post-Golgi carriers being tubular pleomorphic structures that bud from the trans-face of the Golgi. In this review, we present the background studies and highlight the key components of this pathway, we then discuss the machinery implicated in the formation of these carriers, their translocation across the cytosol, and their fusion at the plasma membrane

Rac1 Dynamics in the Human Opportunistic Fungal Pathogen Candida albicans

The small Rho G-protein Rac1 is highly conserved from fungi to humans, with approximately 65% overall sequence identity in Candida albicans. As observed with human Rac1, we show that C. albicans Rac1 can accumulate in the nucleus, and fluorescence recovery after photobleaching (FRAP) together with fluorescence loss in photobleaching (FLIP) studies indicate that this Rho G-protein undergoes nucleo-cytoplasmic shuttling. Analyses of different chimeras revealed that nuclear accumulation of C. albicans Rac1 requires the NLS-motifs at its carboxyl-terminus, which are blocked by prenylation of the adjacent cysteine residue. Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus. Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1. Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans

Direct trafficking pathways from the Golgi apparatus to the plasma membrane.

In eukaryotic cells, protein sorting is a highly regulated mechanism important for many physiological events. After synthesis in the endoplasmic reticulum and trafficking to the Golgi apparatus, proteins sort to many different cellular destinations including the endolysosomal system and the extracellular space. Secreted proteins need to be delivered directly to the cell surface. Sorting of secreted proteins from the Golgi apparatus has been a topic of interest for over thirty years, yet there is still no clear understanding of the machinery that forms the post-Golgi carriers. Most evidence points to these post-Golgi carriers being tubular pleomorphic structures that bud from the trans-face of the Golgi. In this review, we present the background studies and highlight the key components of this pathway, we then discuss the machinery implicated in the formation of these carriers, their translocation across the cytosol, and their fusion at the plasma membrane

The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p.

Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling

Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins

ACBD3 is a protein localised to the Golgi apparatus and recruits other proteins, such as PI4KIIIβ, to the Golgi. However, the mechanism through which ACBD3 itself is recruited to the Golgi is poorly understood. This study demonstrates there are two mechanisms for ACBD3 recruitment to the Golgi. First, we identified that an MWT374-376 motif in the unique region upstream of the GOLD domain in ACBD3 is essential for Golgi localisation. Second, we use unbiased proteomics to demonstrate that ACBD3 interacts with SCFD1, a Sec1/Munc-18 (SM) protein, and a SNARE protein, SEC22B. CRISPR-KO of SCFD1 causes ACBD3 to become cytosolic. We also found that ACBD3 is redundantly recruited to the Golgi apparatus by two golgins: golgin-45 and giantin, which bind to ACBD3 through interaction with the MWT374-376 motif. Taken together, our results suggest that ACBD3 is recruited to the Golgi in a two-step sequential process, with the SCFD1-mediated interaction occurring upstream of the interaction with the golgins

Rac1 dynamics at the plasma membrane depend on its activation state.

<p>(A) Localization of Rac1, activated (Rac1[G12V]) or inactivated (Rac[T17N]) forms. Confocal microscopy images of budding <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPRAC1</i> (PY205), <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPrac1[G12V]</i> (PY209), and <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPrac1[T17N]</i> (PY212) cells were taken; both the maximal projection and central section are shown. (B) Inactivated and activated forms of Rac1 have different plasma membrane distributions. Graph of signal intensity along plasma membrane perimeter of the indicated cells, as shown in the bottom panel of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015400#pone-0015400-g006" target="_blank">Figure 6A</a>. (C) FRAP analysis of cells expressing Rac1 (<i>n</i> = 17), its activated (Rac1[G12V]) (<i>n</i> = 20) or inactivated (Rac[T17N]) (<i>n</i> = 17) forms. FRAP t_½ values (means ± standard deviation) are determined from single-phase exponential curve fit of fluorescence recovery after photobleaching. The two-tailed P-values of the indicated mean FRAP t_½'s (*) are less than 0.006 compared with the FRAP t_½ of GFP-Rac1.</p

Rac1 dynamics in the nucleus depend on its activation state.

<p>(A) Nuclear localization of Rac1 depends on its activation state. DIC and fluorescence images of budding <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPRAC1</i> (PY205), <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPrac1[G12V]</i> (PY209), and <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPrac1[T17N]</i> (PY212) cells after 1 h in the absence of agitation were taken. Bar, 5 µm. (B) Rac1 dynamics at the nucleus depend on its activation state. FRAP analysis of nucleus of <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPRAC1</i> (PY205), (<i>n</i> = 25), <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPrac11[G12V]</i> (PY209), (<i>n</i> = 23), <i>rac1</i>Δ<i>/rac1</i>Δ <i>PADH1GFPrac11[T17N]</i> (PY212) (<i>n</i> = 21), and <i>dck1</i>Δ<i>/dck1</i>Δ <i>PADH1GFPRAC1</i> (PY1265) (<i>n</i> = 30) cells, after 75–90 min in the absence of agitation. FRAP t_½ values (means ± standard deviation) are determined from single-phase exponential curve fits of FRAP intensities, as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015400#pone-0015400-g005" target="_blank">Figure 5A</a>. The two-tailed P-values of the indicated mean FRAP t_½'s (*) are less than 0.0005 compared with the FRAP t_½ of GFP-Rac1.</p