https://orcid.org/0000-0001-7207-4656
Abstract
For proper intracellular vesicle transport, it is essential for transport vesicle membranes to fuse with the appropriate target membranes. Ykt6 is a SNARE protein with functions in diverse vesicle transport pathways, including secretory, endocytotic and autophagic pathways. To exert these functions, the association of Ykt6 with vesicle membranes and the change of its conformation from closed to open play key roles. Recent studies have revealed regulatory mechanisms involved in Ykt6 membrane association and conformation change. When in the cytosol, the vicinal cysteine residues within the C-terminal CCAIM sequence of Ykt6 undergo diprenylation (farnesylation of the distal cysteine residues by farnesyltransferase; this is followed by geranylgeranylation of the proximal cysteine residue by geranylgeranyltransferase-III). Phosphorylation of a serine residue within the SNARE domain triggers the conversion of the Ykt6 conformation from closed to open, allowing Ykt6 membrane association. In this commentary, I briefly summarize and discuss the recently revealed regulatory mechanisms of Ykt6 function by diprenylation and phosphorylation.
Abbreviations
- GGTase
geranylgeranyltransferase
- KO
knockout
- PTAR1
prenyltransferase α subunit repeat-containing 1
- SNARE
soluble N-ethylmaleimide sensitive factor attachment protein receptor
- SNAREs
SNARE proteins
For proper intracellular vesicle transport, it is essential for transport vesicle membranes to fuse with appropriate target membranes and deliver cargo from the donor vesicle to the target organelle (1). The dynamics of vesicle transport are controlled by several types of proteins, such as Rab family small GTPases, tethering factors and soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins. SNARE proteins (SNAREs) form a fusion machinery; their SNARE domain, consisting of about 60 amino acids, mediates the fusion of vesicle membranes with target membranes (2). SNAREs are functionally classified into v-SNAREs, which are associated with vesicles, and t-SNAREs, which are associated with vesicle targets. During the membrane fusion process, v-SNAREs and t-SNAREs on distinct membranes bind to form a trans-SNARE complex. Additionally, SNAREs are structurally classified into Q-SNAREs, having a conserved glutamine (Q) residue, and R-SNAREs, having a conserved arginine (R) residue in the center of the SNARE domain. Q-SNAREs are further classified into Qa-, Qb- and Qc-SNAREs based on the amino acid sequence homology of their SNARE domain (3,4); Qbc-SNAREs, containing two SNARE domains, also exist (4).
Ykt6 is an R-SNARE protein that was first identified in yeast (5) and is known to be highly conserved in organisms, ranging from yeast to humans. Ykt6 is implicated in diverse vesicle transport pathways, including secretory, endocytotic and autophagic pathways (6–10). There are at least two unique features of Ykt6 that are distinct from those of most SNAREs. First, Ykt6 consists of a longin domain at the N-terminus and a SNARE domain at the C-terminus (Fig. 1); however, unlike most SNAREs, which have a C-terminal transmembrane domain for anchoring to membranes, Ykt6 lacks a transmembrane domain (11). Instead, Ykt6 has lipid modifications at its C-terminus, which will be discussed later. The other unique feature of Ykt6 is that its N-terminal longin domain interacts with its C-terminal SNARE domain, allowing it to form a folded/closed conformation (11). In the cytosol, Ykt6 remains inactive by being in the closed conformation, only when it is the open conformation, can it anchor to membranes (12).
A possible model of Ykt6 phosphorylation and diprenylation function in the regulation of Golgi–lysosome transport and autophagy. When in the cytosol, the CCAIM sequence of nascent Ykt6 in closed and open conformations undergoes diprenylation by the catalytic actions of farnesyltransferase, Ras converting enzyme 1, isoprenylcysteine carboxyl methyltransferase and geranygeranyltransferase-III. A serine residue within the SNARE domain of Ykt6 is phosphorylated by protein kinase C iota, which triggers the conversion of Ykt6 from a closed to an open conformation, allowing Ykt6 to anchor to membranes. Phosphorylated Ykt6 can be dephosphorylated by calcineurin. Membrane-associated Ykt6 mediates Golgi–lysosome transport and presumably autophagosome–lysosome fusion. C, cysteine; A, alanine; I, isoleucine; M, methionine; P, phosphorylation, FTase, farnesyltransferase; Rce1, Ras converting enzyme 1; ICMT, isoprenylcysteine carboxyl methyltransferase; GGTase-III, geranygeranyltransferase-III; PKCι, protein kinase C iota.
Post-translational lipid modifications that convert nascent hydrophilic proteins to hydrophobic proteins are pivotal for membrane targeting and binding, because hydrophobic proteins have a higher affinity for membranes. Previous studies have revealed prenylation, which involves attachment of isoprenoid residues to a CAAX motif or a CC, CCXX or CXC motif (C, A and X indicate cysteine, an aliphatic amino acid and any amino acid residue, respectively), of a variety of proteins, such as the Ras superfamily small G proteins (i.e., Ras, Rab and Rho GTPases), subunits of heterotrimeric G proteins and nuclear lamins. Ras GTPases and nuclear lamins are farnesylated, while Rab GTPases, Rho GTPases, and γ subunits of heterotrimeric G proteins are geranylgeranylated. Farnesylation is mediated by farnesyltransferase, which transfers a 15-carbon chain isoprenoid from farnesyl pyrophosphate onto the C-terminal cysteine residue, while geranylgeranylation is mediated by geranylgeranyltransferase-I (GGTase-I), Rab geranylgeranyl transferase/geranylgeranyltransferase-II (GGTase-II) and the recently identified geranylgeranyltransferase-III (GGTase-III) (13,14), which transfer a 20-carbon chain isoprenoid from geranylgeranyl pyrophosphate onto it. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate are produced in the mevalonate pathway by farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, respectively.
Human Ykt6 contains a conserved CAAX prenylation motif with an additional proximal cysteine at the C-terminus (CCAIM) (Fig. 1) (10). The cysteine residue within the CAAX motif is farnesylated by farnesyltransferase (11). It has been reported that farnesylation of nascent Ykt6 existing in both open and closed conformations stabilizes it in a closed conformation (12,15). Once the proximal cysteine residue adjacent to the farnesylated cysteine gets palmitoylated by cellular DHHC palmitoyl-transferases (16), Ykt6 can associate with membranes (11,16). However, in contrast to previous reports, Horiuchi’s group recently provided strong biochemical and structural evidence for geranylgeranylation of the proximal cysteine residue within the CCAIM sequence (13). They reported that Ykt6 can be diprenylated: Ykt6 is first farnesylated and then further geranylgeranylated by GGTase-III. GGTase-III was recently discovered by the Human Genome Project as a novel protein with prenyltransferase homology (14). GGTase-III consists of prenyltransferase α subunit repeat-containing 1 (PTAR1) and the β subunit of Rab geranylgeranyl transferase (13,14), and preferentially recognizes farnesylated Ykt6 (13). Thus, conflicting results have been reported regarding the lipid modification (i.e., palmitoylation or geranylgeranylation) of the Ykt6 CCAIM sequence. The differences between these results are likely due to the presence or absence of the N-terminal tag and/or to an open or closed conformation (13). Ykt6 is the first example of a diprenylated protein that undergoes both farnesylation and geranylgeranylation. Diprenylation is an irreversible modification and is essential but insufficient for the membrane localization of Ykt6 because it was reported that diprenylated Ykt6 was still in a biochemically soluble state (13). Additional modification (i.e., the conformational change of Ykt6 from closed to open) is required for the membrane localization of Ykt6, which will be discussed later.
PTAR1-knockout (KO) cells are a useful research tool to elucidate the role of Ykt6, particularly geranylgeranylation, because PTAR1 exists only in GGTase-III among four prenyltransferases and, currently, only two proteins (Ykt6 and FBXL2), a component of the Skp-Cullin-F box ubiquitin E3 ligase, have been reported to be a substrate for GGTase-III. PTAR1-KO cells, in which Ykt6 is farnesylated, but is not geranylgeranylated, show abnormal Golgi morphology and function, such as delayed intra-Golgi trafficking and impaired protein glycosylation, indicating the significance of the Ykt6 geranylgeranylation (13). Recently, Horiuchi’s group reported, using PTAR1-KO cells, in an article published in The Journal of Biochemistry (17), that the lysosomal hydrolases, cathepsin D and β-hexosaminidase, were missorted at the trans-Golgi network and secreted into the extracellular space. This is the first report showing the essential role of Ykt6 diprenylation in exerting Ykt6 function in lysosomal hydrolase trafficking. Moreover, the autophagy marker LC3B was accumulated in PTAR1-KO cells, suggesting the disturbance of the autophagic protein degradation pathway; however, it remains unclear whether the LC3B accumulation was due to the impairment of autophagosome–lysosome fusion or a defect in the delivery of lysosomal hydrolases to lysosomes. Nevertheless, diprenylated Ykt6, but not monoprenylated Ykt6, is critical for the efficient sorting and trafficking of hydrolases to lysosomes.
It was recently reported that Ykt6 plays a key role in the fusion of autophagosomes with lysosomes, the final step in macroautophagy (8,9). The results shown in the previous paper (13) also suggested an essential role of Ykt6 geranylgeranylation for Ykt6 membrane association. However, geranylgeranylation is not a trigger for the conformational change of Ykt6 from closed to open (15), an essential step in the membrane association of Ykt6. It was recently reported that a conserved serine residue (Ser174) within the SNARE domain is phosphorylated by atypical protein kinase C iota (18) and that this phosphorylation, regulated by Ca2+ signalling, triggers the conversion of Ykt6 from a closed to an open conformation, allowing its anchorage to membranes (Fig. 1) (18,19), although it remains unclear how the activity of Ca2+-independent atypical protein kinase C iota, which lacks a C2 domain that binds Ca2+, is regulated by Ca2+ signalling. Phosphorylation of diprenylated Ykt6 at Ser174 stabilizes the open conformation and enables it to bind to a membrane (18). Membrane-bound, phosphorylated Ykt6 can be dephosphorylated by calcineurin (18), and dephosphorylated Ykt6 mediates Golgi–lysosome transport and presumably autophagosome–lysosome fusion. Intriguingly, it was reported that in yeast two conserved Ser182 and Ser183 within the SNARE domain of Ykt6 are phosphorylated by Atg1 kinase, which renders Ykt6 inactive and prevents autophagosome–vacuole fusion (20,21). Thus, phosphorylation/dephosphorylation may play roles in the regulation of the membrane association of Ykt6. However, when interpreting research results using N-terminally tagged Ykt6, we need to consider carefully whether these Ykt6 mutants reflect the behaviour of diprenylated Ykt6 because it was reported that N-terminal tag on Ykt6 interfered with its geranylgeranylation by preventing the binding of GGTase-III to Ykt6 (13), and N-terminally GFP-tagged Ykt6 was not functional in yeast (20). Whether additional regulatory mechanisms of the membrane association of Ykt6 exist and how membrane-associated Ykt6 returns to the cytosol after membrane fusion remain to be elucidated. Because previous studies have implicated Ykt6 in various pathological conditions, including cancer (22–24) and Parkinson’s disease (18,25), elucidating regulatory mechanisms and functional roles of Ykt6 post-translational modifications will open new avenues for our understanding of these diseases and the development of therapeutic strategies for them.
Acknowledgement
This work was supported by JSPS KAKENHI [grant number 20K07192].
Conflict of Interest
The author declares no conflict of interest.
