Curr. Issues Mol. Biol. (2020) 35: 99-108. Role of Ubiquitin and SUMO in Intracellular Trafcking Maria Sundvall1,2,3* 1Institute of Biomedicine, University of Turku, Turku, Finland. 2Western Cancer Centre FICAN West, Turku University Hospital, Turku, Finland. 3Department of Oncology and Radiotherapy, University of Turku, Turku, Finland. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.035.099 Abstract behaviour. In addition to e.g. chemical groups, Precise location of proteins at a given time within such as phosphate groups, proteins can be modi- a cell is essential to convey specifc signals and fed by small polypeptides, such as ubiquitin and result in a relevant functional outcome. Small SUMO. Ubiquitin and SUMO share a similar ubiquitin-like modifcations, such as ubiquitin three-dimensional structure and are principally and SUMO, represent a delicate and diverse way covalently linked at lysine (K) residues of sub- to transiently regulate intracellular trafcking strate proteins by a similar conjugation pathway events of existing proteins in cells. Trafcking (Hershko et al., 1998; Hay, 2013). Unlike ubiq- of multiple proteins is controlled reversibly by uitin, SUMO is preferentially atached at SUMO ubiquitin and/or SUMO directly or indirectly via consensus sites ΨKxE in substrates Ψ = hydropho- regulation of transport machinery components. bic residue with high preference for I or V, x = any Regulation is dynamic and multilayered, involv- amino acid) under steady state conditions, but ing active crosstalk and interdependence between under stress conditions in particular more non- post-translational modifcations. However, in consensus sites are SUMOylated (Hendriks et al., most cases regulation appears very complex, and 2016). Mammals express ubiquitously SUMO1, the mechanistic details regarding how ubiquitin SUMO2 and SUMO3, whereas SUMO4 and and SUMO control protein location in cells are SUMO5 are only expressed in some tissues and not yet fully understood. Moreover, most of the their functional role is unclear (Pichler et al., fndings still lack in vivo evidence in multicellular 2017). SUMO2 and SUMO3 are nearly identi- organisms. cal and resemble approximately 50% to SUMO1 (Geiss-Friedlander et al., 2007). Precursors of ubiquitin and SUMO are pro- Posttranslational modifcations cessed to mature forms prior to conjugation and in regulation of cellular activated by an E1 enzyme in a reaction depend- processes ing on ATP. Subsequently ubiquitin and SUMO are transferred to an E2 enzyme, and E3 ligase General principles of ubiquitination alone or with E2 ligates them to substrates by and SUMOylation an isopeptide bond (Hershko et al., 1998; Hay, Postranslational modifcation (PTM) of pro- 2013). Modifcation is reversible and specifc teins is a powerful, fast and ofen transient way proteases can cleave ubiquitin and SUMO from to control the fate of existing proteins and cell substrates (Williamson et al., 2013; Kunz et al., caister.com/cimb 99 Curr. Issues Mol. Biol. Vol. 35 304 | Sundvall 2018). Proteins can be tagged by a single ubiqui- E1 (Sae1/Aos1–Sae2/Uba2) and E2 (Ubc9) tin or SUMO either at one or at multiple lysines. are known to be involved in SUMO conjugation In addition, modifcations can exist as chains. (Hay, 2013). Tree classes of SUMO E3s have Ubiquitin contains seven internal lysine residues been widely accepted and characterized including (K6, K11, K27, K29, K33, K48, K63) that are SP-RING Siz/PIAS ligases, RanBP2 and ZNF451 involved in the formation of ubiquitin-ubiquitin ligases (Geiss-Friedlander et al., 2007; Rytinki et polymers, known as polyubiquitin chains (Pickart al., 2009; Cappadocia et al., 2015). Both E2 and et al., 2004; Heride et al., 2014). In addition, the E3 can select substrates for SUMOylation, and linkage between the amino-terminal amino group spatial and temporal regulation of co-localization of methionine on a ubiquitin can be conjugated appears integral for substrate selection (Pichler with a target protein and the carboxy-terminal et al., 2017). Cysteine proteases of the sentrin- carboxy group of the incoming ubiquitin for specifc protease (SENP) family members reverse linear chains (Walczak et al., 2012). SUMOs SUMO conjugation in mammals (Kunz et al., can also form polymeric chains through internal 2018). Moreover, desumoylating isopeptides 1 lysine residues (Geiss-Friedlander et al., 2007). and 2 and ubiquitin-specifc protease-like 1 can Monoubiquitination and diferent chain types deSUMOylate proteins (Shin et al., 2012; Schulz et determine the fate of the modifed protein (Piper al., 2012). Whereas ubiquitin machinery is widely et al., 2014). For example, K48 ubiquitin chains expressed within a cell, components of SUMO are considered classical signals for proteasomal conjugation pathway mainly localize at the nuclear degradation and K63 ubiquitin chains are linked pores and nucleus. Tus, most of SUMOylated to trafcking and DNA damage response (Pick- substrates are nuclear proteins, although SUMO art et al., 2004). Recently basic principles of the modifed proteins outside of nucleus exist (Geiss- feld have been challenged by e.g. discoveries of Friedlander et al., 2007). mixed polyubiquitin chains and ubiquitination of Ubiquitination is regulated by extracellular non-lysine residues (Piper et al., 2014). Less is stimuli including growth factors and cytokines, known regarding the consequences of atachment stress and cell cycle changes (Pickart et al., 2004; of either single or many SUMO moieties. SUMO Heride et al., 2014). Diferent types of stress stimuli chains are at least implicated in recruitment of such as heat shock, hypoxia, reactive oxygen spe- SUMO-targeted ubiquitin ligases (Lallemand- cies, DNA damage and proteotoxic stress regulate Breitenbach et al., 2008). the activity of SUMOylation machinery (Hieta- kangas et al., 2003; Shao et al., 2004; Bossis et al., Components, regulation and function 2006; Galanty et al., 2009; Morris et al., 2009; of ubiquitin and SUMO machinery Seifert et al., 2015). Both ubiquitin and SUMO Multiple enzymes involved in ubiquitin conjuga- modifcations can alter the function, activity, loca- tion have been recognized. Te human ubiquitin tion and stability of their targets. Ubiquitin and machinery comprises a network including two SUMO are recognized by either ubiquitin-binding ubiquitin E1 enzymes, approximately 40 ubiquitin domains (UBD) or sumo-interacting domains E2s, and more than 600 E3 ubiquitin ligases in (SIM), respectively, and serve as platforms for the human genome (Heride et al., 2014). Tree non-covalent protein–protein interactions (Seet major types of E3 ligases are really interesting et al., 2006). Tese domains have been identi- new gene (RING) type E3s comprising most fed in hundreds of proteins. PTMs are also of human E3s, homologous to E6-AP carboxyl involved in regulation of conjugation specifcity terminus (HECT) and RING-between-RING and activation (Pichler et al., 2017). For example, (RBR) E3s ligases (Deshaies et al., 2009; Rotin SUMO-targeted ubiquitin ligase (STUbL) RNF4 et al., 2009; Wenzel et al., 2012). Ubiquitin is contains multiple SIMs and a RING-domain to cleaved by approximately 100 deubiquitinating bind SUMOylated proteins and an E2 ubiquitin- enzymes (DUBs) (Williamson et al., 2013; Heride conjugation enzyme (Sun et al., 2007; Tatham et et al., 2014). Intriguingly, only one heterodimeric al., 2008). caister.com/cimb 100 Curr. Issues Mol. Biol. Vol. 35 Ubiquitin and SUMO in Intracellular Trafcking | 305 Ubiquitin and SUMO as signals epsin and endosomal sortin complex required for regulating membrane trafcking transport (ESCRT) (Piper et al., 2014). Ubiquitin and endocytosis is also indirectly involved in control of endocytosis as components of endocytic machinery are actively General principles of membrane regulated by ubiquitination (Piper et al., 2014). protein trafcking and endocytosis in SUMOylation has been implicated in the cells regulation of endocytic processes, although Internalization and endocytosis of cell surface when compared to ubiquitin the evidence is less proteins including diferent receptors ofen occurs extensive, and more work is needed to make general via the clathrin-dependent endocytic pathway. conclusions. Nevertheless, endocytosis of kainate Cell surface receptors are clustered to pits coated receptor GluR6 is regulated by SUMOylation with clathrin that pinch of of the membrane and non-SUMOylated mutant of GluR6, GluR6- forming vesicles and early endosomes. From early K886R, is endocytosis-impaired due to unknown endosomes cargo can be directed back to plasma mechanisms (Martin et al., 2007). SUMOylation membrane via recycling endosomes or destined to of Smoothened (Smo) promotes its localization lysosomal degradation via late endosomes and mul- at the cell surface (Ma et al., 2016). Intriguingly, tivesicular bodies (Mellman and Yarden, 2013). mechanistically SUMO interferes with efcient Smo Also, other types of endocytic routes exist, includ- ubiquitination by recruiting deubiquitinase UBPY/ ing cholesterol-rich membrane structures, such USP8 in a SIM-dependent manner (Ma et al., 2016). as lipid rafs and caveolae (Barbieri et al., 2016). SUMOylation regulates also cell surface expression Several adaptor proteins and PTMs control these and activity of VEGFR2 receptor tyrosine kinase processes (Piper et al., 2014). Membrane protein (Zhou et al., 2018). VEGFR2–SUMO1 fusion trafcking and endocytosis system are tightly con- protein but not SUMOylation defective mutant nected