Sandbox 122

Identification of the cargo and Transport through the NPC

In eukaryotes, proteins must be transported in and out of the nucleus. This nucleocytoplasmic transport of proteins across the nuclear envelope must occur through the gateway of the NPC. The NPC is a large structure consisting of 456 constituent binding proteins called nucleoporins (Nups).1 Movement through the NPC is facilitated transport that relies on interaction with specific Nups. Importins and exportins are proteins that aid this facilitated transport by both binding to a specific cargo to be transported and interacting with specific Nups located in the central channel of the NPC.2

Karyopherinβ is a group of proteins that is composed of both importins and exportins. Importins are proteins that carry cargos into the nucleus while exportins serve the opposite function. As of today, twenty different Kapβs have been identified. Each of these Kapβs is capable of recognizing and transporting a specific group of cargos. In order to bind to its cargo a Kapβ has to recognize a Nuclear Localizaton or Export Signal (NLS or NES) is located in the polypeptide chain of the cargo. These signals can vary from 7 amino acids to longer than 100 amino acids in length.

An importin, such as Kapβ2, binds to a specific cargo by recognition of an NLS and carries the cargo through the NPC by interacting with intrinsically disordered Nups called FG-Nups. FG-Nups line the passageway of the NPC and contain repeats of phenylalanine and glycine. These unstructured FG-Nups form a low-density cloud within the central channel extending from the cytoplasm to the nucleoplasm. The cloud acts as an effective exclusion filter for those particles that do not contain FG repeat binding sites. This is referred to as the zone of selectivity.

Structure of Kapβ2 Kapβ2 is a superhelix comprised of 20 HEAT repeats (the name HEAT derives from Huntington, Elongation factor 3 A subunit of protein phosphatase 2A and Tor1 kinase), each of which consists of two The electrostatic potential of the internal surface of Kapβ2 superhelix at the C-terminal arch is negative.

HEAT repeats and form the of Kapβ2 cargos while repeats constitute the RanGTPase binding site.3 RanGTPase, a small 216-residue protein, is found more frequently in the nucleus and enables cargos to be released from Kapβ2.

Kapβ2 Cargo Binding and Conformational Change4

The located on Kapβ2 cargos are named the PY- NLS and they bind to the C-terminal arch of Kapβ2.

Recognition of the PY-NLS by Kapβ2 follows certain guidelines:

(i) PY-NLS, when not bound to Kapβ2, lacks a secondary structure.

(ii) PY-NLS has an overall positive charge allowing for electrostatic compatibility with Kapβ2.

(iii) General sequence for the PY-NLS is either a hydrophobic or basic motif at the N-terminus and a R-X2-5-P-Y motif at the C-terminus.

The contains a hydrophobic rather than a basic N- terminal motif. Hydrophobic interactions at the N- terminal motif of the PY- NLS include: of the NLS with Trp730 and Ile773 of Kapβ2. Interactions at the C- terminal R-X2-5-P-Y motif of the NLS include:

of the NLS with Glu509 and Asp543 of Kapβ2; of the NLS with Ala380, Ala 381, Ala 384, Leu419, Ile457, Trp460 and Arg464 of Kapβ2.

Upon binding Kapβ2, the NLS gains structure, conforms to and makes contact with the internal surface of the Kapβ2 C-terminal arch.

How does Kapβ2 pass through the NPC?

Once the cargo binds to Kapβ2, the complex travels through the NPC by interactions with the FG-Nups.