Subba Rao Gangi Setty

Golgi targeting of ARF-like GTPase Arl3p requires its Nα-acetylation and the integral membrane protein Sys1p

Myristoylation of ARF family GTPases is required for their association with Golgi and endosomal membranes, where they regulate protein sorting and the lipid composition of these organelles. The Golgi-localized ARF-like GTPase Arl3p/ARP lacks a myristoylation signal, indicating that its targeting mechanism is distinct from myristoylated ARFs. We demonstrate that acetylation of the N-terminal methionine of Arl3p requires the NatC Nα-acetyltransferase and that this modification is required for its Golgi localization. Chemical crosslinking and fluorescence microscopy experiments demonstrate that localization of Arl3p also requires Sys1p, a Golgi-localized integral membrane protein, which may serve as a receptor for acetylated Arl3p.

Golgi recruitment of GRIP domain proteins by Arf-like GTPase 1 is regulated by Arf-like GTPase 3.

Golgins are Golgi-localized proteins present in all molecularly characterized eukaryotes that function in Golgi transport and maintenance of Golgi structure. Some peripheral membrane Golgins, including the yeast Imh1 protein, contain the recently described GRIP domain that can independently mediate Golgi localization by an unknown mechanism. To identify candidate Golgi receptors for GRIP domain proteins, a collection of Saccharomyces cerevisiae deletion mutants was visually screened by using yeast, mouse, and human GFP-GRIP domain fusion proteins for defects in Golgi localization. GFP-GRIP reporters were localized to the cytosol in cells lacking either of two ARF-like (ARL) GTPases, Arl1p and Arl3p. In vitro binding experiments demonstrated that activated Arl1p-GTP binds specifically and directly to the Imh1p GRIP domain. Arl1p colocalized with Imh1p-GRIP at the Golgi, and Golgi localization of Arl1p was regulated by the GTPase cycle of Arl3p. These results suggest a cascade in which the GTPase cycle of Arl3p regulates Golgi localization of Arl1p, which in turn binds to the GRIP domain of Imh1p and recruits it to the Golgi. The similar requirements for localization of GRIP domains from yeast, mouse, and human when expressed in yeast, and the presence of Arl1p and Arl3p homologs in these species, suggest that this is an evolutionarily conserved mechanism.

ESCRT-I function is required for Tyrp1 transport from early endosomes to the melanosome limiting membrane

Melanosomes are lysosome‐related organelles that coexist with lysosomes within melanocytes. The pathways by which melanosomal proteins are diverted from endocytic organelles toward melanosomes are incompletely defined. In melanocytes from mouse models of Hermansky‐Pudlak syndrome that lack BLOC‐1, melanosomal proteins such as tyrosinase‐related protein 1 (Tyrp1) accumulate in early endosomes. Whether this accumulation represents an anomalous pathway or an arrested normal intermediate in melanosome protein trafficking is not clear. Here, we show that early endosomes are requisite intermediates in the trafficking of Tyrp1 from the Golgi to late stage melanosomes in normal melanocytic cells. Kinetic analyses show that very little newly synthesized Tyrp1 traverses the cell surface and that internalized Tyrp1 is inefficiently sorted to melanosomes. Nevertheless, nearly all Tyrp1 traverse early endosomes since it becomes trapped within enlarged, modified endosomes upon overexpression of Hrs. Although Tyrp1 localization is not affected by Hrs depletion, depletion of the ESCRT‐I component, Tsg101, or inhibition of ESCRT function by dominant‐negative approaches results in a dramatic redistribution of Tyrp1 to aberrant endosomal membranes that are largely distinct from those harboring traditional ESCRT‐dependent, ubiquitylated cargoes such as MART‐1. The lysosomal protein content of some of these membranes and the lack of Tyrp1 recycling to the plasma membrane in Tsg101‐depleted cells suggests that ESCRT‐I functions downstream of BLOC‐1. Our data delineate a novel pathway for Tyrp1 trafficking and illustrate a requirement for ESCRT‐I function in controlling protein sorting from vacuolar endosomes to the limiting membrane of a lysosome‐related organelle.

Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes.

OCA2 is used as a model melanosome cargo protein to define primary sequence elements required for acidic dileucine–motif binding to adaptors AP-1 and AP-3. OCA2 must bind to AP-3 for melanosome localization. BLOC-1 is also required and thus can cooperate with either adaptor for cargo delivery to lysosome-related organelles.

BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery

Quantitative analyses of melanosome cargo localization and trafficking and of endosomal membrane dynamics in immortalized melanocytes from mouse Hermansky–Pudlak syndrome models show that BLOC-2 functions to specify the delivery of recycling endosomal cargo transport intermediates to maturing melanosomes.